Antiviral oligonucleotides

ABSTRACT

Random sequence oligonucleotides that have antiviral activity are described, along with their use as antiviral agents. In many cases, the oligonucleotides are greater than 40 nucleotides in length. Also described are methods for the prophylaxis or treatment of a viral infection in a human or animal, and a method for the prophylaxis treatment of cancer caused by oncoviruses in a human or animal. The methods typically involve administering to a human or animal in need of such treatment, a pharmacologically acceptable, therapeutically effective amount of at least oligonucleotide that does not act by a sequence complementary mode of action.

RELATED APPLICATIONS

This application is a continuation-in-part of Juteau & Vaillant, PCTapplication serial number not yet assigned, filed Sep. 11, 2003,entitled ANTIVIRAL OLIGONUCLEOTIDES, and claims the benefit of Vaillant& Juteau, U.S. Provisional Appl. 60/430,934, filed Dec. 5, 2002 and ofVaillant & Juteau, U.S. Provisional Appl. 60/410,264, filed Sep. 13,2002, all of which are incorporated herein by reference in theirentireties, including drawings.

FIELD OF THE INVENTION

The present invention relates to oligonucleotides having antiviralactivities and their use as therapeutic agents in viral infectionscaused by human and animal viruses and in cancers caused by oncogeneviruses and in other diseases whose etiology is viral-based.

BACKGROUND OF THE INVENTION

The following discussion is provided solely to assist the understandingof the reader, and does not constitute an admission that any of theinformation discussed or references cited constitute prior art to thepresent invention.

Many important infectious diseases afflicting mankind are caused byviruses. Many of these diseases, including rabies, smallpox,poliomyelitis, hepatitis, yellow fever, immune deficiencies and variousencephalitic diseases, are frequently fatal. Others are significant inthat they are highly contagious and create acute discomfort such asinfluenza, measles, mumps and chickenpox, as well as respiratory orgastrointestinal disorders. Others such as rubella and cytomegaloviruscan cause congenital abnormalities. Finally there are viruses, known asoncoviruses, which can cause cancer in humans and animals.

Among viruses, the family of Herpesviridae is of great interest. TheHerpesviridae are a ubiquitous class of icoshedral, double stranded DNAviruses. Of over 100 characterized members of Herpesviridae (HHV), onlyeight infect humans. The best known among these are Herpes simplex type1 (HSV-1), Herpes simplex type 2 (HSV-2), Varicella zoster (chicken poxor shingles), cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Theprevalence of Herpes viruses in humans is high, affecting at least onethird of the worldwide population; and in the United States, 70-80% ofthe population have some kind of Herpes infection. While the pathologyof Herpes infections are usually not dangerous, as in the case of HSV-1which usually only causes short lived lesions around the mouth and face,these viruses are also known to be the cause of more dangerous symptoms,which vary from genital ulcers and discharge to fetal infections whichcan lead to encephalitis (15% mortality) or disseminated infection (40%mortality).

Herpes viruses are highly disseminated in nature and highly pathogenicfor man. For example, Epstein-Barr virus (EBV) is known to causeinfectious mononucleosis in late childhood or adolescence or in youngadults. The hallmarks of acute infectious mononucleosis are sore throat,fever, headache, lymphadenopathy, enlarged tonsils and atypical,dividing lymphocytes in the peripheral blood. Other manifestationsfrequently include mild hepatitis, splenomegaly and encephalitis. EBV isalso associated with two forms of cancer: Burkitt's lymphoma (BL) andthe nasopharyngeal carcinoma (NPC). In endemic areas of equatorialAfrica, BL is the most common childhood malignancy, accounting forapproximately 80% of cancers in children. While moderately observed inNorth American Caucasians, NPC is one of the most common cancers inSouthern China with age incidence of 25 to 55 years. EBV, like thecytomegalovirus, is also associated with post-transplantlymphoproliferative disease, which is a potentially fatal complicationof chronic immunosuppression following solid organ or bone marrowtransplantation.

Other diseases are also associated with HSV, including skin and eyeinfections, for example, chorioretinitis or keratoconjunctivitis.Approximately 300,000 cases of HSV infections of the eye are diagnosedyearly in the United States.

AIDS (acquired immunodeficiency syndrome) is caused by the humanimmunodeficiency virus (HIV). By killing or damaging cells of the body'simmune system, HIV progressively destroys the body's ability to fightinfections and certain cancers. There are currently approximately 42million people living with HIV/AIDS worldwide. A total of 3.1 millionpeople died of HIV/AIDS related causes in 2002. The ultimate goal ofanti-HIV drug therapy is to prevent the virus from reproducing anddamaging the immune system. Although substantial progress has been madeover the past fifteen years in the fight against HIV, a cure stilleludes medical science. Today, physicians have more than a dozenantiretroviral agents in three different drug classes to manage thedisease. Typically, drugs from two or three classes are prescribed in avariety of combinations known as HAART (Highly Active AntiRetroviralTreatment). HAART therapies typically comprise two nucleoside reversetranscriptase inhibitors drugs with a third drug, either a proteaseinhibitor or a non-nucleoside reverse transcriptase inhibitor. Clinicalstudies have shown that HAART is the most effective means of reducingviral loads and minimizing the likelihood of drug resistance.

While HAART has been shown to reduce the amount of HIV in the body,commonly known as viral load, tens of thousands of patients encountersignificant problems with this therapy. Some side effects are seriousand include abnormal fat metabolism, kidney stones, and heart disease.Other side effects such as nausea, vomiting, and insomnia are lessserious, but still problematic for HIV patients that need chronic drugtherapy for a lifetime.

Currently approved anti-HIV drugs work by entering an HIV infected CD4+T cell and blocking the function of a viral enzyme, either the reversetranscriptase or a protease. HIV needs both of these enzymes in order toreproduce. However, HIV frequently mutates and become resistant,rendering reverse transcriptase or protease inhibitor drugs ineffective.Once resistance occurs, viral loads increase and dictate the need toswitch the ineffective agent for another antiretroviral agent.Unfortunately, when a virus becomes resistant to one drug in a class,other drugs in that class may become less effective. This phenomenonknown as cross-resistance, occurs because many anti-HIV drugs work insimilar manners. The occurrence of drug cross-resistance is highlyundesirable because it reduces the available number of treatment optionsfor patients.

There is therefore a great need for the development of other antiviralagents effective against HIV that work through other mechanisms ofaction against which the virus has not developed resistance. This isbecoming especially important in view of recent data showing that 1 outof 10 patients newly diagnosed with HIV in Europe, is infected with astrain of HIV already resistant to at least one of the approved drug onthe market.

Respiratory syncytial virus (RSV) causes upper and lower respiratorytract infections. It is a negative-sense, enveloped RNA virus and ishighly infectious. It commonly affects young children and is the mostcommon cause of lower respiratory tract illness in infants. RSVinfections are usually associated with moderate-to-severe cold-likesymptoms. However, severe lower respiratory tract disease may occur atany age, especially in elderly or immunocompromised patients. Childrenwith severe infections may require oxygen therapy and, in certain cases,mechanical ventilation. According to the American Medical Association,an increasing number of children are being hospitalized forbronchiolitis, often caused by RSV infection. RSV infections alsoaccount for approximately one-third of community-associated respiratoryvirus infections in patients in bone marrow transplant centers. In theelderly population, RSV infection has been recently recognized to bevery similar in severity to influenza virus infection.

Influenza (INF), also known as the flu, is a contagious disease that iscaused by the influenza virus. It attacks the respiratory tract inhumans (nose, throat, and lungs). An average of about 36,000 people peryear in the United States die from influenza, and 114,000 per yearrequire hospitalization as a result of influenza.

In all infectious diseases, the efficacy of a given therapy oftendepends on the host immune response. This is particularly true forherpes viruses, where the ability of all herpes viruses to establishlatent infections results in an extremely high incidence of reactivatedinfections in immunocompromised patients. In renal transplantrecipients, 40% to 70% reactivate latent HSV infections, and 80% to 100%reactivate CMV infections. Such viral reactivations have also beenobserved with AIDS patients.

The hepatitis B virus (HBV) is a DNA virus that belongs to theHepadnaviridae family of viruses. HBV causes hepatitis B in humans. Itis estimated that 2 billion people have been infected (1 out of 3people) in the world. About 350 million people remain chronicallyinfected and an estimated 1 million people die each year from hepatitisB and its complications. HBV can cause lifelong infection, cirrhosis ofthe liver, liver cancer, liver failure, and death. The virus istransmitted through blood and bodily fluids. This can occur throughdirect blood-to-blood contact, unprotected sex, use of unsterileneedles, and from an infected woman to her newborn during the deliveryprocess. Most healthy adults (90%) who are infected will recover anddevelop protective antibodies against future hepatitis B infections. Asmall number (5-10%) will be unable to get rid of the virus and willdevelop chronic infections while 90% of infants and up to 50% of youngchildren develop chronic infections when infected with the virus.Alpha-interferon is the most frequent type of treatment used.Significant side effects are related to this treatment includingflu-like symptoms, depression, rashes, other reactions and abnormalblood counts. Another treatment option includes 3TC which also has manyside effects associated with its use. In the last few years, there hasbeen an increasing number of reports showing that patients treated with3TC are developing resistant strains of HBV. This is especiallyproblematic in the population of patients who are co-infected with HBVand HIV. There is clearly an urgent need to develop new antiviraltherapies against this virus.

Hepatitis C virus (HCV) infection is the most common chronic bloodborneinfection in the United States where the number of infected patientslikely exceeds 4 million. This common viral infection is a leading causeof cirrhosis and liver cancer, and is now the leading reason for livertransplantation in the United States. Recovery from infection isuncommon, and about 85 percent of infected patients become chroniccarriers of the virus and 10 to 20 percent develop cirrhosis. It isestimated that there are currently 170 million people worldwide who arechronic carriers. According to the Centers for Disease Control andPrevention, chronic hepatitis C causes between 8,000 and 10,000 deathsand leads to about 1,000 liver transplants in the United States aloneeach year. There is no vaccine available for hepatitis C. Prolongedtherapy with interferon alpha, or the combination of interferon withRibavirin, is effective in only about 40 percent of patients and causessignificant side effects.

Today, the therapeutic outlook for viral infections in general is notfavourable. In general, therapies for viruses have mediocre efficaciesand are associated with strong side effects which either prevent theadministration of an effective dosage or prevent long term treatment.Three clinical situations which exemplify these problems areherpesviridae, HIV and RSV infections.

In the case of herpesviridae, there are five major treatments currentlyapproved for use in the clinic: idoxuridine, vidarabine, acyclovir,foscarnet and ganciclovir. While having limited efficacy, thesetreatments are also fraught with side effects. Allergic reactions havebeen reported in 35% of patients treated with idoxuridine, vidarabinecan result in gastrointestional disturbances in 15% of patients andacyclovir, foscarnet and ganciclovir, being nucleoside analogs, affectDNA replication in host cells. In the case of ganciclovir, neutropeniaand thrombocytopenia are reported in 40% of AIDS patients treated withthis drug.

While there are many different drugs currently available for thetreatment of HIV infections, all of these are associated with sideeffects potent enough to require extensive supplemental medication togive patients a reasonable quality of life. The additional problem ofdrug resistant strains of HIV (a problem also found in herpesviridaeinfections) usually requires periodic changing of the treatment cocktailand in some cases, makes the infection extremely difficult to treat.

The treatment of RSV infections in young infants is another example ofthe urgent need for new drug development. In this case, the usual lineof treatment is to deliver Ribavirin by inhalation using asmall-particule aerosol in an isolation tent. Not only is Ribavirin onlymildly effective, but its uses is associated with significant sideeffects. In addition, the potential release of the drug has caused greatconcern in hospital personnel because of the known teratogenicity ofRibavirin.

It is clear that for any new emerging antiviral drug being developed, itwould be highly desirable to incorporate the three following features:1—improved efficacy; 2—reduced risks of side effects and 3—a mechanismof action which is difficult for the virus to overcome by mutation.

Several attempts to inhibit particular viruses by various antisenseapproaches have been made.

Zamecnik et al. have used ONs specifically targeted to the reversetranscriptase primer site and to splice donor/acceptor sites (Zamecnik,et al (1986) Proc. Natl. Acad. Sci. USA 83: 4143-) (Goodchild & Zamecnik(1989) U.S. Pat. No. 4,806,463).

Crooke and coworkers. (Crooke et al. (1992) Antimicrob. AgentsChemother. 36: 527-532) described an antisense against HSV-1.

Draper et al. (1993) (U.S. Pat. No. 5,248,670) have reported antisenseoligonucleotides having anti-HSV activity containing the Cat sequenceand hybridizing to the HSV-1 genes UL13, UL39 and UL40.

Kean et al. (Biochemistry (1995) 34: 14617-14620) have tested antisensemethylphosphonate oligomers as anti-HSV agents.

Peyman et al. (Biol Chem Hoppe Seyler (1995) Mar; 376: 195-198) havereported testing specific antisense oligonucleotides directed againstthe IE110 and the UL30 mRNA of HSV-1 for their antiviral properties.

Oligonucleotides or oligonucleotide analogs targeting CMV mRNAs codingfor IE1, IE2 or DNA polymerase were reported by Anderson et al (1997)(U.S. Pat. No. 5,591,720).

Hanecak et al (1999) (U.S. Pat. No. 5,952,490) have described modifiedoligonucleotides having a conserved G quartet sequence and a sufficientnumber of flanking nucleotides to significantly inhibit the activity ofa virus such as HSV-1.

Jairath et al (Antiviral Res. (1997) 33: 201-213) have reportedantisense oligonucleotides against RSV.

Torrence et al (1999) (U.S. Pat. No. 5,998,602) have reported compoundscomprising an antisense component complementary to a single strandedportion of the RSV antigenomic strand (the mRNA strand), a linker and aoligonucleotide activator of RNase L.

Qi et al. (Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi (2000) 14:253-256) have reported testing antisense PS-ODNs in Coxsackie virus B3.

International publication WO9203051 (Roizman and Maxwell) describesmethylphosphonate antisense oligomers which are complementary to vitalregions of HSV viral genome or mRNA transcripts thereof which exhibitantiviral activity.

Guanosine/thymidine or guanosine-rich phophorothioateoligodeoxynucleotides (GT-PS-ODNs) have been reported to have antiviralactivity. The article stated that “several different PS-containingGT-rich ODNs (B106-140, I100-12, and G106-57) all 26 or 27 nt in length,were just as effective at reducing HIV-2 titers as GT-rich ODNsconsisting of 36 (B106-96, B106-97) or 45 nt (Table 4).” (Fennewald etal., Antiviral Res. (1995) 26: 37-54).

In U.S. Pat. No. 6,184,369, anti-HIV, anti-HSV, and anti-CMVoligonucleotides containing a high percentage of guanosine bases aredescribed. In preferred embodiments, the oligonucleotide has a threedimensional structure and this structure is stabilized by guanosinetetrads. In a further embodiment, the oligonucleotide compositions ofthe invention have two or more runs of two contiguous deoxyguanosinesThe patent claims a G-rich ODN that includes at least two G residues inat least two positions.

Cohen et al. (U.S. Pat. Nos. 5,264,423 and 5,276,019) described theinhibition of replication of HIV, and more particularly to PS-ODNanalogs that can be used to prevent replication of foreign nucleic acidsin the presence of normal living cells. Cohen et al describe antiviralactivity of antisense PS-ODNs specific to a viral sequence. They alsodescribe testing polyA, polyT and polyC PS-ODN sequences of 14, 18, 21and 28-mers and indicate an antiviral effect of those PS-ODNs.

Matsukura et al. (Matsukura et al (1987) Proc Natl Acad Sci USA 84:7706-7710) later published the result described in Cohen et al, USpatents above.

Gao et al (Gao et al (1989) J Biol Chem 264: 11521-11526), describe theinhibition of replication of HSV-2, by PS-ODNs by testing of polyA,polyT and polyC PS-ODN sequences in sizes of 7, 15, 21 and 28nucleotides.

Archambault, Stein and Cohen (Archambault et al (1994) Arch Virol 139:97109) report that a PS-ODN polyc of 28 nucleotides is not effectiveagainst HSV-1.

Stein et al (Stein et al. (1989) AIDS Res Hum Retrovir 5: 639-646),published results concerning additional data on anti-HIV ODNs, generallyof 21-28 nucleotides in length.

Marshal et al. (Marshall et al. (1992) Proc. Natl. Acad. Sci. USA 89:6265-6269) describe anti-HIV-1 effect of phosphorothioate andphosphorothioate poly-C oligos of 4-28 nucleotides in length.

Stein & Cheng (Stein et al. (1993) Science 261: 1004-1012), in a reviewarticle, mention the antiviral activity of non specific ODNs of 28nucleotides, stating that “the anti-HIV properties of PS oligos aresignificantly influenced by non-sequence-specific effects, that is, theinhibitory effect is independent of the base sequence.”

In a review article Lebedeva & Stein (Lebedeva et al (2001) Annul RevPharmacol 41: 403-419) report a variety of non-specific protein bindingactivity of PS-ODNs, including viral proteins. They state that “thesemolecules are highly biologically active, and it is often relativelyeasy to mistake artifact for antisense”.

Rein et al. (U.S. Pat. No. 6,316,190) reported a GT rich ON decoy linkedto a fusion partner and binding to the HIV nucleocapsid, which can beused as an antiviral compound. Similarly, Campbell et al. (Campbell etal (1999) J. Virol. 73: 2270-2279) reported PO-ODN with a TGTGT motifbinding specifically to the nucleocapsid of HIV but with no referencesto an antiviral activity.

Feng at al. (Feng et al. (2002) J. Virol. 76: 11757-11762) describedA(n) and TG(n) PO-ODNs binding to the recombinant HIV nucleocapsid butwith no data nor references to an anti-HIV activity.

Antisense ODNs developed as anticancer agents, antiviral agents, or totreat others diseases are typically approximately 20 nucleotides inlength. In a review article (Stein, C A, (2001) J. Clin. Invest. 108:641-644), it is affirmed that “the length of an antisenseoligonucleotide must be optimized: If the antisense oligonucleotide iseither too long or too short, an element of specificity is lost. At thepresent time, the optimal length for an antisense oligonucleotide seemsto be roughly 16-20 nucleotides”. Similarly, in another review article(Crooke, ST (2000) Methods Enzymol. 313: 3-45) it is stated that“Compared to RNA and RNA duplex formation, a phosphorothioateoligodeoxynucleotide has a T_(m) approximately −2.20 lower per unit.This means that to be effective in vitro, phosphorothioateoligodeoxynucleotides must typically be 17- to −20-mer in length . . .”.

Caruthers and co-workers (Marshall et al. (1992) Proc. Natl. Acad. Sci.USA 89: 6265-6269) reported anti-HIV activity of phosphorodithioate ODNs(PS2-ODNs) for a 12mer polycytidine-PS2-ODN and for a 14mer PS2-ODN. Noother sizes were tested for anti-HIV activity. They also reported theinhibition of HIV reverse transcriptase (RT) for 12, 14, 20 and 28merpolycytidine-PS2-ODNs. Later, (Marshal et al (1993) Science 259:1564-1570) reported results showing sequence specific inhibition of theHIV RT. The same group published data for PS2-ODNs in several patents.In U.S. Pat. Nos. 5,218,103 and 5,684,148, PS2-ODN structure andsynthesis is described. In U.S. Pat. Nos. 5,452,496, 5,278,302, and5,695,979 inhibition of HIV RT is described for PS2-ODNs not longer than15 bases. In U.S. Pat. Nos. 5,750,666 and 5,602,244, antisense activityof PS2-ODNs is described.

SUMMARY OF THE INVENTION

The present invention involves the discovery that oligonucleotides(ONs), e.g., oligodeoxynucleotides (ODNs), can have a broadlyapplicable, non-sequence complementary antiviral activity. Thus, it isnot necessary for the oligonucleotide to be complementary to any viralsequence or to have a particular distribution of nucleotides in order tohave antiviral activity. Such an oligonucleotide can even be prepared asa randomer, such that there will be at most a few copies of anyparticular sequence in a preparation, e.g., in a 15 micromol randomerpreparation 32 or more nucleotides in length.

In addition, the inventors discovered that different lengtholigonucleotides have varying antiviral effect, and further that thelength of antiviral oligonucleotide that produces maximal antiviraleffect is in the range of 40-120 nucleotides. In view of the presentdiscoveries concerning antiviral properties of oligonucleotides, thisinvention provides oligonucleotide antiviral agents that can haveactivity against numerous different viruses, and can even be selected asbroad-spectrum antiviral agents. Such antiviral agents are particularlyadvantageous in view of the limited antiviral therapeutic optionscurrently available.

Therefore, the ONs, e.g., ODNs, of the present invention are useful intherapy for treating or preventing viral infections or for treating orpreventing tumors or cancers induced by viruses, such as oncoviruses(e.g., retroviruses, papillomaviruses, and herpesviruses), and intreating or preventing other diseases whose etiology is viral-based.Such treatments are applicable to many types of patients and treatments,including, for example, the prophylaxis or treatment of viral infectionsin immunosuppressed human and animal patients.

In a first aspect, the invention provides an antiviral oligonucleotideformulation that includes at least one antiviral oligonucleotide, e.g.,at least 6 nucleotides in length, adapted for use as an antiviral agent,where the antiviral activity of the oligonucleotide occurs principallyby a non-sequence complementary mode of action. Such a formulation caninclude a mix of different oligonucleotides, e.g., at least 2, 3, 5, 10,50, 100, or even more.

As used herein in connection with oligonucleotides or other materials,the term “antiviral” refers to an effect of the presence of theoligonucleotides or other material in inhibiting production of viralparticles, i.e., reducing the number of infectious viral particlesformed, in a system otherwise suitable for formation of infectious viralparticles for at least one virus. In certain embodiments of the presentinvention, the antiviral oligonucleotides will have antiviral activityagainst multiple different virus.

The term “antiviral oligonucleotide formulation” refers to a preparationthat includes at least one antiviral oligonucleotide that is adapted foruse as an antiviral agent. The formulation includes the oligonucleotideor oligonucleotides, and can contain other materials that do notinterfere with use as an antiviral agent in vivo. Such other materialscan include without restriction diluents, excipients, carrier materials,and/or other antiviral materials.

As used herein, the term “pharmaceutical composition” refers to anantiviral oligonucleotide formulation that includes a physiologically orpharmaceutically acceptable carrier or excipient. Such compositions canalso include other components that do not make the compositionunsuitable for administration to a desired subject, e.g., a human.

In the context of the present invention, unless specifically limited theterm “oligonucleotide (ON)” means oligodeoxynucleotide (ODN) oroligodeoxyribonucleotide or oligoribonucleotide. Thus, “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. Examples of modifications thatcan be used are described herein. Oligonucleotides that include backboneand/or other modifications can also be referred to as oligonucleosides.

As used in connection with an antiviral formulation, pharmaceuticalcomposition, or other material, the phrase “adapted for use as anantiviral agent” indicates that the material exhibits an antiviraleffect and does not include any component or material that makes itunsuitable for use in inhibiting viral production in an in vivo system,e.g., for administering to a subject such as a human subject.

As used herein in connection with antiviral action of a material, thephrase “non-sequence complementary mode of action” indicates that themechanism by which the material exhibits an antiviral effect is not dueto hybridization of complementary nucleic acid sequences, e.g., anantisense effect. Conversely, a “sequence complementary mode of action”means that the antiviral effect of a material involves hybridization ofcomplementary nucleic acid sequences. Thus, indicating that theantiviral activity of a material is “not primarily due to a sequencecomplementary mode of action” means that the the activity of theoligonucleotide satisfies at least one of the 4 tests provided herein(see Example, 10) for determining whether the antiviral activity is “notprimarily due to a sequence complementary mode of action”. In particularembodiments, the oligonucleotide satisfies test 1, test 2, test 3, ortest 4; the oligonucleotide satisfies a combination of two of the tests,i.e., tests 1 & 2; tests 1 & 3; tests 1 & 4, tests 2 & 3, tests 2 & 4,or tests 3 & 4; the oligonucleotide satisfies a combination of 3 of thetests, i.e., tests 1, 2, and 3, tests 1, 2, and 4, tests 1, 3, and 4, ortests 2, 3, and 4; the oligonucleotide satisifies all of tests 1, 2, 3,and 4.

As used herein in connection with administration of an antiviralmaterial, the term “subject” refers to a living higher organism,including, for example, animals such as mammals, e.g., humans, non-humanprimates, bovines, porcines, ovines, equines, dogs, and cats; birds; andplants, e.g., fruit trees.

A related aspect concerns an antiviral oligonucleotide randomerformulation, where the antiviral activity of the randomer occursprincipally by a non-sequence complementary mode of action. Such arandomer formulation can, for example, include a mixture of randomers ofdifferent lengths, e.g., at least 2, 3, 5, 10, or more differentlengths.

As used herein in connection with oligonucleotide sequences, the term“random” characterizes a sequence or an ON that is not complementary toa viral mRNA, and which is selected to not form hairpins and not to havepalindromic sequences contained therein. When the term “random” is usedin the context of antiviral activity of an oligonucleotide toward aparticular virus, it implies the absence of complementarity to a viralmRNA of that particular virus. The absence of complementarity may bebroader, e.g., for a plurality of viruses, for viruses from a particularviral family, or for infectious human viruses.

In the present application, the term “randomer” is intended to mean asingle stranded DNA having a wobble (N) at every position, such asNNNNNNNNNN. Each base is synthesized as a wobble such that this ONactually exists as a population of different randomly generatedsequences of the same size.

In another aspect, the invention provides an oligonucleotide havingantiviral activity against a target virus, where the oligonucleotide isat least 29 nucleotides in length (or in particular embodiments, atleast 30, 32, 34, 36, 38, 40, 46, 50, 60, 70, 80, 90, 100, 110, or 120nucleotides in length) and the sequence of the oligonucleotide is notcomplementary to any portion of the genome sequence of the target virus.

In another aspect, the invention provides an oligonucleotideformulation, containing at least one oligonucleotide having antiviralactivity against a target virus, where the oligonucleotide is at least 6nucleotides in length (in particular embodiments, at least 29, 30, 32,34, 36, 38, 40, 46, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides inlength) and the sequence of oligonucleotide is less than 70%complementary to any portion of the genomic nucleic acid sequence of thetarget virus and does not consist essentially of polyA, polyc, polyG,polyT, Gquartet, or a TG-rich sequence. In particular embodiments, theoligonucleotide has less than 65%, 60%, 55%, 50%, 80% 90%, 95%, or 100%complementarity to any portion of the genomic nucleic acid sequence ofthe target virus.

As used in connection with the present oligos, the term “TG-rich”indicates that the sequence of the antiviral oligonucleotide consists ofat least 70 percent T and G nucleotides, or if so specified, at least80, 90, or 95% T and G, or even 100%.

Related aspects concern isolated, purified or enriched antiviraloligonucleotides as described herein, e.g., as described for antiviraloligonucleotide formulations, as well as other oligonucleotidepreparations, e.g., preparations suitable for in vitro use.

Antiviral oligonucleotides useful in the present invention can be ofvarious lengths, e.g., at least 6, 10, 14, 15, 20, 25, 28, 29, 30, 35,38, 40, 46, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or morenucleotides in length. Likewise, the oligonucleotide can be in a range,e.g., a range defined by taking any two of the preceding listed valuesas inclusive end points of the range, for example 10-20, 20-40, 30-50,40-60, 40-80, 60-120, and 80-120 nucleotides. In particular embodiments,a minimum length or length range is combined with any other of theoligonucleotide specifications listed herein for the present antiviraloligonucleotides.

The antiviral nucleotide can include various modifications, e.g.,stabilizing modifications, and thus can include at least onemodification in the phosphodiester linkage and/or on the sugar, and/oron the base. For example, the oligonucleotide can include one or morephosphorothioate linkages, phosphorodithioate linkages, and/ormethylphosphonate linkages; modifications at the 2′-position of thesugar, such as 2′-O-methyl modifications, 2′-amino modifications,2′-halo modifications such as 2′-fluoro; acyclic nucleotide analogs, andcan also include at least one phosphodiester linkage. Othermodifications are also known in the art and can be used. In oligos thatcontain 2′-O-methyl modifications, the oligo should not have 2′-O-methylmodifications throughout, as current results suggest that such oligos donot have suitable activity. In particular embodiments, theoligonucleotide has modified linkages throughout, e.g.,phosphorothioate; has a 3′- and/or 5′-cap; includes a terminal 3′-5′linkage; the oligonucleotide is or includes a concatemer consisting oftwo or more oligonucleotide sequences joined by a linker(s).

In particular embodiments, the oligonucleotide binds to one or moreviral proteins; the sequence of the oligonucleotide (or a portionthereof, e.g., at least ½) is derived from a viral genome; the activityof an oligonucleotide with a sequence derived from a viral genome is notsuperior to a randomer oligonucleotide or a random oligonucleotide ofthe same length; the oligonucleotide includes a portion complementary toa viral sequence and a portion not complementary to a viral sequence;the sequence of the oligonucleotide is derived from a viral packagingsequence or other viral sequence involved in an aptameric interaction;unless otherwise indicated, the sequence of the oligonucleotide includesA(x), C(x), G(x), T(x), AC(x), AG(x), AT(x), CG(x), CT(x), or GT(x),where x is 2, 3, 4, 5, 6, . . . 60 . . . 120 (in particular embodimentsthe oligonucleotide is at least 29, 30, 32, 34, 36, 38, 40, 46, 50, 60,70, 80, 90, 100, 110, or 120 nucleotides in length or the length of thespecified repeat sequence is at least a length just specified); theoligonucleotide is single stranded (RNA or DNA); the oligonucleotide isdouble stranded (RNA or DNA); the oligonucleotide includes at least oneGquartet or CpG portion; the oligonucleotide includes a portioncomplementary to a viral mRNA and is at least 29, 37, or 38 nucleotidesin length (or other length as specified above); the oligonucleotideincludes at least one non-Watson-Crick oligonucleotide and/or at leastone nucleotide that participates in non-Watson-Crick binding withanother nucleotide; the oligonucleotide is a random oligonucleotide, theoligonucleotide is a randomer or includes a randomer portion, e.g., arandomer portion that has a length as specified above foroligonucleotide length; the oligonucleotide is linked or conjugated atone or more nucleotide residues to a molecule that modifies thecharacteristics of the oligonucleotide, e.g. to provide higher stability(such as stability in serum or stability in a particular solution),lower serum interaction, higher cellular uptake, higher viral proteininteraction, improved ability to be formulated for delivery, adetectable signal, improved pharmacokinetic properties, specific tissuedistribution, and/or lower toxicity.

Oligonucleotides can also be used in combinations, e.g., as a mixture.Such combinations or mixtures can include, for example, at least 2, 4,10, 100, 1000, 10000, 100,000, 1,000,000, or more differentoligonucleotides. Such combinations or mixtures can, for example, bedifferent sequences and/or different lengths and/or differentmodifications and/or different linked or conjugated molecules. Inparticular embodiments of such combinations or mixtures, a plurality ofoligonucleotides have a minimum length or are in a length range asspecified above for oligonucleotides. In particular embodiments of suchcombinations or mixtures, at least one, a plurality, or each of theoligonucleotides can have any of the other properties specified hereinfor individual antiviral oligonucleoties (which can also be in anyconsistent combination).

The phrase “derived from a viral genome” indicates that a particularsequence has a nucleotide base sequence that has at least 70% identityto a viral genomic nucleotide sequence or its complement (e.g., is thesame as or complementary to such viral genomic sequence), or is acorresponding RNA sequence. In particular embodiments of the presentinvention, the term indicates that the sequence is at least 70%identical to a viral genomic sequence of the particular virus againstwhich the oligonucleotide is directed, or to its complementary sequence.In particular embodiments, the identity is at least 80, 90, 95, 98, 99,or 100%.

The invention also provides an antiviral pharmaceutical composition thatincludes a therapeutically effective amount of a pharmacologicallyacceptable, antiviral oligonucleotide at least 6 nucleotides in length(or other length as listed herein), where the antiviral activity of theoligonucleotide occurs principally by a non-sequence complementary modeof action, and a pharmaceutically acceptable carrier. In particularembodiments, the oligonucleotide or a combination or mixture ofoligonucleotides is as specified above for individual oligonucleotidesor combinations or mixtures of oligonucleotides. In particularembodiments, the pharmaceutical compositions are approved foradministration to a human, or a non-human animal such as a non-humanprimate.

In particular embodiments, the pharmaceutical composition is adapted forthe treatment, control, or prevention of a disease with a viraletiology; adapted for treatment, control, or prevention of a priondisease; is adapted for delivery by intraocular administration, oralingestion, enteric administration, inhalation, cutaneous, subcutaneous,intramuscular, intraperitoneal, intrathecal, intratracheal, orintravenous injection, or topical administration. In particularembodiments, the composition includes a delivery system, e.g., targetedto specific cells or tissues; a liposomal formulation, another antiviraldrug, e.g., a non-nucleotide antiviral polymer, an antisense molecule,an siRNA, or a small molecule drug.

In particular embodiments, the antiviral oligonucleotide,oligonucleotide preparation, oligonucleotide formulation, or antiviralpharmaceutical composition has an IC50 for a target virus (e.g., any ofparticular viruses or viruses in a groups of viruses as indicatedherein) of 0.50, 0.20, 0.10, 0.09. 0.08, 0.07, 0.75, 0.06, 0.05, 0.045,0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or 0.01 μM or less.

In particular embodiments of formulations, pharmaceutical compositions,and methods for prophylaxis or treatment, the composition or formulationis adapted for treatment, control, or prevention of a disease with viraletiology; is adapted for the treatment, control or prevention of a priondisease; is adapted for delivery by a mode selected from the groupconsisting of intraocular, oral ingestion, enterally, inhalation, orcutaneous, subcutaneous, intramuscular, or intravenous injectiondelivery; further comprises a delivery system, which can include or beassociated with a molecule increasing affinity with specific cells;further comprises at least one other antiviral drug in combination;and/or further comprises an antiviral polymer in combination.

As used herein in connection with antiviral oligonucleotides andformulations, and the like, in reference to a particular virus or groupof viruses the term “targeted” indicates that the oligonucleotide isselected to inhibit that virus or group of viruses. As used inconnection with a particular tissue or cell type, the term indicatesthat the oligonucleotide, formulation, or delivery system is selectedsuch that the oligonucleotide is preferentially present and/orpreferentially exhibits an antiviral effect in or proximal to theparticular tissue or cell type.

As used herein, the term “delivery system” refers to a component orcomponents that, when combined with an oligonucleotide as describedherein, increases the amount of the oligonucleotide that contacts theintended location in vivo, and/or extends the duration of its presenceat the target, e.g., by at least 20, 50, or 100%, or even more ascompared to the amount and/or duration in the absence of the deliverysystem, and/or prevents or reduces interactions that cause side effects.

As used herein in connection with antiviral agents and other drugs ortest compounds, the term “small molecule” means that the molecularweight of the molecule is 1500 daltons or less. In some cases, themolecular weight is 1000, 800, 600, 500, or 400 daltons or less.

In another aspect, the invention provides a kit that includes at leastone antiviral oligonucleotide or oligonucleotide formulation in alabeled package, where the antiviral activity of the oligonucleotideoccurs principally by a non-sequence complementary mode of action andthe label on the package indicates that the antiviral oligonucleotidecan be used against at least one virus.

In particular embodiments the kit includes a pharmaceutical compositionthat includes at least one antiviral oligonucletide as described herein;the antiviral oligonucleotide is adapted for in vivo use in an animaland/or the label indicates that the oligonucleotide or composition isacceptable and/or approved for use in an animal; the animal is a mammal,such as human, or a non-human mammal such as bovine, porcine, aruminant, ovine, or equine; the animal is a non-human animal; the kit isapproved by a regulatory agency such as the U.S. Food and DrugAdministration or equivalent agency for use in an animal, e.g., a human.

In another aspect, the invention provides a method for selecting anantiviral oligonucleotide, e.g, a non-sequence complementary antiviraloligonucleotide, for use as an antiviral agent. The method involvessynthesizing a plurality of different random oligonucleotides, testingthe oligonucleotides for activity in inhibiting the ability of a virusto produce infectious virions, and selecting an oligonucleotide having apharmaceutically acceptable level of activity for use as an antiviralagent.

In particular embodiments, the different random oligonucleotidescomprises randomers of different lengths; the random oligonucleotidescan have different sequences or can have sequence in common, such as thesequence of the shortest oligos of the plurality; and/or the differentrandom oligonucleotides comprise a plurality of oligonucleotidescomprising a randomer segment at least nucleotides in length or thedifferent random oligonucleotides include a plurality of randomers ofdifferent lengths. Other oligonucleotides, e.g., as described herein forantiviral oligonucleotides, can be tested in a particular system.

In yet another aspect, the invention provides a method for theprophylaxis or treatment of a viral infection in a subject byadministering to a subject in need of such treatment a therapeuticallyeffective amount of at least one pharmacologically acceptableoligonucleotide as described herein, e.g., a non-sequence complementaryoligonucleotide at least 6 nucleotides in length, or an antiviralpharmaceutical composition or formulation containing sucholigonucleotide. In particular embodiments, the virus can be any ofthose listed herein as suitable for inhibition using the presentinvention; the infection is related to a disease or condition indicatedherein as related to a viral infection; the subject is a type of subjectas indicated herein, e.g., human, non-human animal, non-human mammal,plant, and the like; the treatment is for a viral disease or diseasewith a viral etiology, e.g., a disease as indicated in the Backgroundherein.

In particular embodiments, an antiviral oligonucleotide (oroligonucleotide formulation or pharmaceutical composition) as describedherein is administered; administration is a method as described herein;a delivery system or method as described herein is used; the viralinfection is of a DNA virus or an RNA virus; the virus is aparvoviridae, papovaviridae, adenoviridae, herpesviridae, poxyiridae,hepadnaviridae, or papillomaviridae; the virus is a arenaviridae,bunyaviridae, calciviridae, coronaviridae, filoviridae, flaviridae,orthomyxoviridae, paramyxoviridae, picornaviridae, reoviridae,rhabdoviridae, retroviridae, or togaviridae; the herpesviridae virus isEBV, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, or HHV-8; the virus is HIV-1or HIV-2; the virus is RSV; the virus is an influenza virus, e.g.,influenza A; the virus is HBV; the virus is smallpox virus or vacciniavirus; the virus is a coronavirus; the virus is SARS virus; the virus isWest Nile Virus; the virus is a hantavirus; the virus is a parainfluenzavirus; the virus is coxsackievirus; the virus is rhinovirus; the virusis yellow fever virus; the virus is dengue virus; the virus is hepatitisC virus; the virus is Ebola virus; the virus is Marburg virus.

Similarly, in a related aspect, the invention provides a method for theprophylactic treatment of cancer caused by oncoviruses in a human oranimal by administering to a human or animal in need of such treatment,a pharmacologically acceptable, therapeutically effective amount of atleast one random oligonucleotide of at least 6 nucleotides in length (oranother length as described herein), or a formulation or pharmaceuticalcomposition containing such oligonucleotide.

In particular embodiments, the oligonucleotide(s) is as described hereinfor the present invention, e.g., having a length as described herein; amethod of administration as described herein is used; a delivery systemas described herein is used.

The term “therapeutically effective amount” refers to an amount that issufficient to effect a therapeutically or prophylactically significantreduction in production of infectious virus particles when administeredto a typical subject of the intended type. In aspects involvingadministration of an antiviral oligonucleotide to a subject, typicallythe oligonucleotide, formulation, or composition should be administeredin a therapeutically effective amount.

In another aspect, the discovery that non-sequence complementaryinteractions produce effective antiviral activity provides a method ofscreening to identify a compound that alters binding of anoligonucleotide to a viral component, such as one or more viral proteins(e.g., extracted or purified from a viral culture of infected hostorganisms, or produced by recombinant methods). For example, the methodcan involve determining whether a test compound reduces the binding ofoligonucleotide to one or more viral components.

As used herein, the term “screening” refers to assaying a plurality ofcompounds to determine if they possess a desired property. The pluralityof compounds can, for example, be at least 10, 100, 1000, 10,000 or moretest compounds.

In particular embodiments, any of a variety of assay formats anddetection methods can be used to identify such alteration in binding,e.g., by contacting the oligonucleotide with the viral component(s) inthe presence and absence of a compound(s) to be screened (e.g., inseparate reactions) and determining whether a difference occurs inbinding of the oligo the viral component(s) in the presence of thecompound compared to the absence of the compound. The presence of such adifference is indicative that the compound alters the binding of therandom oligonucleotide to the viral component. Alternatively, acompetitive displacement can be used, such that oligonucleotide is boundto the viral component and displacement by added test compound isdetermined, or conversely test compound is bound and displacement byadded oligonucleotide is determined.

In particular embodiments, the oligonucleotide is as described hereinfor antiviral oligonucleotides; the oligonucleotide is at least 6, 8,10, 15, 20, 25, 29, 30, 32, 34, 36, 38, 40, 46, 50, 60, 70, 80, 90, 100,110, or 120 nucleotides in length or at least another length specifiedherein for the antiviral oligonucleotides, or is in a range defined bytaking any two of the preceding values as inclusive endpoints of therange; the test compound(s) is a small molecule; the test compound has amolecular weight of less than 400, 500, 600, 800, 1000, 1500, 2000,2500, or 3000 daltons, or is in a range defined by taking any two of thepreceding values as inclusive endpoints of the range; the viral extractor component is from a virus as listed herein; at least 100, 1000,10,000, 20,000, 50,000, or 100,000 compounds are screened; theoligonucleotide has an IC50 of equal to or less than 0.500, 0.200,0.100, 0.075, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or0.01 μM.

As used herein, the term “viral component” refers to a product encodedby a virus or produced by infected host cells as a consequence of theviral infection. Such components can include proteins as well as otherbiomolecules. Such viral components, can, for example, be obtained fromviral cultures, infected host organisms, e.g., animals and plants, orcan be produced from viral sequences in recombinant systems (prokaryotesand eukaryotes), as well synthetic proteins having amino acid sequencescorresponding to viral encoded proteins. The term “viral cultureextract” refers to an extract from cells infected by a virus that willinclude virus-specific products. Similarly, a “viral protein” refers toa virus-specific protein, usually encoded by a virus, but can also beencoded at least in part by host sequences as a consequence of the viralinfection.

In a related aspect, the invention provides an antiviral compoundidentified by the preceding method, e.g., a novel antiviral compound.

In a further aspect, the invention provides a method for purifyingoligonucleotides binding to at least one viral component from a pool ofoligonucleotides by contacting the pool with at least one viralcomponent, e.g., bound to a stationary phase medium, and collectingoligonucleotides that bind to the viral component(s). Generally, thecollecting involves displacing the oligonucleotides from the viralcomponent(s). The method can also involve sequencing and/or testingantiviral activity of collected oligonucleotides (i.e., oligonucleotidesthat bound to viral protein).

In particular embodiments, the bound oligonucleotides of the pool aredisplaced from the stationary phase medium by any appropriate method,e.g., using an ionic displacer, and displaced oligonucleotides arecollected. Typically for the various methods of displacement, thedisplacement can be performed in increasing stringent manner (e.g., withan increasing concentration of displacing agent, such as a saltconcentration, so that there is a stepped or continuous gradient), suchthat oligonucleotides are displaced generally in order of increasedbinding affinity. In many cases, a low stringency wash will be performedto remove weakly bound oligonucleotides, and one or more fractions willbe collected containing displaced, tighter binding oligonucleotides. Insome cases, it will be desired to select fractions that contain verytightly binding oligonucleotides (e.g., oligonucleotides in fractionsresulting from displacement by the more stringent displacementconditions) for further use.

Similarly, the invention provides a method for enrichingoligonucleotides from a pool of oligonucleotides binding to at least oneviral component, by contacting the pool with one or more viral proteins,and amplifying oligonucieotides bound to the viral proteins to providean enriched oligonucleotide pool. The contacting and amplifying can beperformed in multiple rounds, e.g., at least 1, 2, 3, 4, 5, 10, or moreadditional times using the enriched oligonucleotide pool from thepreceding round as the pool of oligonucleotides for the next round. Themethod can also involve sequencing and testing antiviral activity ofoligonucleotides in the enriched oligonucleotide pool following one ormore rounds of contacting and amplifying.

The method can involve displacing oligonucleotides from the viralcomponent (e.g., viral protein bound to a solid phase medium) with anyof a variety of techniques, such as those described above, e.g., using adisplacement agent. As indicated above, it can be advantageous to selectthe tighter binding oligonucleotides for further use, e.g., in furtherrounds of binding and amplifying. The method can further involveselecting one or more enriched oligonucleotides, e.g., high affinityoligonucleotides, for further use. In particular embodiments, theselection can include eliminating oligonucleotides that have sequencescomplementary to host genomic sequences (e.g., human) for a particularvirus of interest. Such elimination can involve comparing theoligonucleotide sequence(s) with sequences from the particular host in asequence database(s), e.g., using a sequence alignment program (e.g., aBLAST search), and eliminating those oligonucleotides that havesequences identical or with a particular level of identity to a hostsequence. Eliminating such host complementary sequences and/or selectingone or more oligonucleotides that are not complementary to hostsequences can also be done for the other aspects of the presentinvention.

In the preceding methods for identifying, purifying, or enrichingoligonucleotides, the oligonucleotides can be of types as describedherein. The above methods are advantageous for identifying, purifying orenriching high affinity oligonucleotides, e.g., from an oligonucleotiderandomer preparation.

In a related aspect, the invention concerns an antiviral oligonucleotidepreparation that includes one or more oligonucleotides identified usinga method of any of the preceding methods for identifying, obtaining, orpurifying antiviral oligonucleotides from an initial oligonucleotidepool, where the oligonucleotides in the oligonucleotide preparationexhibit higher mean binding affinity with one or more viral proteinsthan the mean binding affinity of oligonucletides in the initialoligonucleotide pool.

In particular embodiments, the mean binding affinity of theoligonucleotides is at least two-fold, 3-fold, 5-fold, 10-fold, 20-fold,50-fold, or 100-fold greater than the mean binding affinity ofoligonucleotides in the initial oligonucleotide pool, or even more; themedian of binding affinity is at least two-fold, 3-fold, 5-fold,10-fold, 20-fold, 50-fold, or 100-fold greater relative to the median ofthe binding affinity of the initial oligo pool, where median refers tothe middle value.

In yet another aspect, the invention provides an antiviral polymer mixthat includes at least one antiviral oligonucleotide and at least onenon-nucleotide antiviral polymer. In particular embodiments, theoligonucleotide is as described herein for antiviral oligonucleotidesand/or the antiviral polymer is as described herein or otherwise knownin the art or subsequently identified.

In yet another aspect, the invention provides an oligonucleotiderandomer, where the randomer is at least 6 nucleotides in length. Inparticular embodiments the randomer has a length as specified above forantiviral oligonucleotides; the randomer includes at least onephosphorothioate linkage, the randomer includes at least onephosphorodithioate linkage or other modification as listed herein; therandomer oligonucleotides include at least one non-randomer segment(such as a segment complementary to a selected virus nucleic acidsequence), which can have a length as specified above foroligonucleotides; the randomer is in a preparation or pool ofpreparations containing at least 5, 10, 15, 20, 50, 100, 200, 500, or700 micromol, 1, 5, 7, 10, 20, 50, 100, 200, 500, or 700 mmol, or 1 moleof randomer, or a range defined by taking any two different values fromthe preceding as inclusive end points, or is synthesized at one of thelisted scales or scale ranges.

Likewise, the invention provides a method for preparing antiviralrandomers, by synthesizing at least one randomer, e.g., a randomer asdescribed above.

As indicated above, for any aspect involving a viral infection or riskof viral infection or targeting to a particular virus, in particularembodiments the virus is as listed above.

The expression “human and animal viruses” is intended to include,without limitation, DNA and RNA viruses in general. DNA viruses include,for example, parvoviridae, papovaviridae, adenoviridae, herpesviridae,poxyiridae, hepadnaviridae, and papillomaviridae. RNA viruses include,for example, arenaviridae, bunyaviridae, calciviridae, coronaviridae,filoviridae, flaviridae, orthomyxoviridae, paramyxoviridae,picornaviridae, reoviridae, rhabdoviridae, retroviridae, or togaviridae.

In connection with modifying characteristics of an oligonucleotide bylinking or conjugating with another molecule or moiety, themodifications in the characteristics are evaluated relative to the sameoligonucleotide without the linked or conjugated molecule or moiety.

Additional embodiments will be apparent from the Detailed Descriptionand from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). Infected cells are treated with increasing concentrationsof REP 1001 (a), REP 2001 (b) or REP 3007 (c). IC₅₀ values calculatedfrom linear regressions are reported in each graph.

FIG. 2. Relationship between PS-ODN size and IC₅₀ against HSV-1. IC₅₀values from FIG. 1 are plotted against the specific size of each PS-ODNtested in FIG. 1.

FIG. 3. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). Infected cells are treated with increasing concentrationsof REP 2001 (a), REP 2002 (b) or REP 3003 (c), REP 2004 (d), REP 2005(e), REP 2006 (f) and Acyclovir (g). IC₅₀ values calculated from linearregressions are reported in each graph.

FIG. 4. Relationship between PS-ODN size and IC₅₀ against HSV-1. IC₅₀values from FIG. 3 are plotted against the specific size of each PS-ODNtested in FIG. 3 which showed anti-HSV-1 activity. The IC₅₀ forAcyclovir is indicated for reference to a clinical correlate.

FIG. 5. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). A broad range of PS-ODN randomer sizes were tested inincreasing concentrations; REP 2003 (a), REP 2009 (b), REP 2010 (c), REP2011 (d), REP 2012 (e), REP 2004 (f), REP 2006 (g), REP 2007 (h) and REP2008 (i). IC₅₀ values calculated from linear regressions are reported ineach graph.

FIG. 6. UV backshadowing of PS-ODN randomers tested in FIG. 5 separatedby acrylamide gel electrophoresis.

FIG. 7. Relationship between PS-ODN randomer size and IC₅₀ againstHSV-1. IC₅₀ values from FIG. 5 are plotted against the specific size ofeach PS-ODN tested in FIG. 5 which showed anti-HSV-1 activity.

FIG. 8. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). Umodified ODNs, PS-ODNs with a random sequence and PS-ODNstargeting the start codon of HSV-1 IE110 were tested in increasingconcentrations. REP 2013 (a), REP 2014 (b), REP 2015 (c), REP 2016 (d),REP 2017 (e), REP 2018 (f), REP 2019 (g), REP 2020 (h) and REP 2021 (i).IC₅₀ values calculated from linear regressions are reported in eachgraph.

FIG. 9. UV backshadowing of PS-ODN randomers tested in FIG. 8 separatedby acrylamide gel electrophoresis.

FIG. 10. Relationship between PS-ODN randomer, PS-ODN random sequence,PS-ODN HSV-1 IE110 sequence and IC₅₀ against HSV-1. IC₅₀ values fromFIG. 8 are plotted against the specific size of each PS-ODN tested inFIG. 8 which showed anti-HSV-1 activity. Additional IC₅₀ values fromFIG. 5 are included for comparison against PS-ODN randomers.

FIG. 11. Plaque reduction assay conducted in VERO cells using HSV-1(strain KOS). A PS-ODN having 2-O methyl modifications to the 4 ribosesugars at each end of the oligo (REP 2024, [a]); a ODN havingmethylphosphonate modifications to the 4 ester linkages at each end ofthe oligo (REP 2026 [b]); and RNA PS-ODNs 20 bases (REP2059 [c]) and 30bases (REP2060 [d]) in length were tested in increasing concentrations.IC₅₀ values calculated from linear regressions are reported in eachgraph.

FIG. 12. Plaque reduction assay conducted in human fibroblast cellsusing HSV-2 (strain MS2). Infected cells are treated with increasingconcentrations of REP 1001 (a), REP 2001 (b) or REP 3007 (c). IC₅₀values calculated from linear regressions are reported in each graph.

FIG. 13. Relationship between PS-ODN size and IC₅₀ against HSV-2. IC₅₀values from FIG. 12 are plotted against the specific size of each PS-ODNtested in FIG. 12.

FIG. 14. Plaque reduction assay conducted in VERO cells using HSV-2(strain MS2). Infected cells are treated with increasing concentrationsof REP 2001 (a), REP 2002 (b) or REP 2003 (c), REP 2004 (d), REP 2005(e), REP 2006 (f) and acyclovir (g). IC₅₀ values calculated from linearregressions are reported in each graph.

FIG. 15. Relationship between PS-ODN size and IC₅₀ against HSV-2. IC₅₀values from FIG. 14 are plotted against the specific size of each PS-ODNtested in FIG. 14 which showed anti-HSV-2 activity. The IC₅₀ foracyclovir is provided for reference to a clinical correlate.

FIG. 16. Plaque reduction assay conducted in VERO cells using CMV(strain AD169). Infected cells are treated with increasingconcentrations of REP 2004 (a) or REP 2006 (b). IC₅₀ values calculatedfrom linear regressions are reported in each graph. The relationshipbetween PS-ODN size and IC₅₀ against CMV is plotted in (c). IC₅₀ valuesfrom FIGS. (a) and (b) are plotted against the specific size of eachPS-ODN tested.

FIG. 17. Plaque reduction assay conducted in VERO cells using CMV(strain AD169). Three clinical CMV therapies were tested: Gancyclovir(a), Foscarnet (b) and Cidofovir (c). A broad range of PS-ODN randomersizes were also tested in increasing concentrations; REP 2003 (d), REP2004 (e), REP 2006 (f) and REP 2007 (g). Finally, REP 2036 (Vitravene)was tested as synthesized in house (h) and as commercially available(i). IC₅₀ values calculated from linear regressions are reported in eachgraph.

FIG. 18. Relationship between PS-ODN size and IC₅₀ against CMV. IC₅₀values from FIG. 17 are plotted against the specific size of each PS-ODNtested in FIG. 17 which showed anti-CMV activity.

FIG. 19. CPE assay conducted in MT4 cells using HIV-1 (strain NL4-3).Infected cells are treated with increasing concentrations of REP 2004(a) or REP 2006 (b). IC₅₀ values calculated from linear regressions arereported in each graph. Cytotoxicity profiles in uninfected MT4 cellsare presented for REP 2004 (c) and REP 2006 (d).

FIG. 20. Relationship between PS-ODN size and IC₅₀ against HIV-1. IC₅₀values from FIG. 1 are plotted against the specific size of each PS-ODNtested in FIG. 1.

FIG. 21. Replication assay conducted in 293A cells using recombinantwild type HIV-1 NL4-3 (strain CNDO). Infected cells are treated withincreasing concentrations of Amprenavir (a), Indinavir (b), Lopinavir(c), Saquinavir (d), REP 2003 (e), REP 2004 (f), REP 2006 (g) and REP2007 (h). Both curves (black and dotted lines) represent dose responsecurves against strain CNDO.

FIG. 22. (a) IC50 values from FIG. 21 and (b), relationship betweenPS-ODN size and IC₅₀ against recombinant HIV-1. IC₅₀ values from (a) areplotted against the specific size of each PS-ODN tested in FIG. 21.

FIG. 23. Replication assay conducted in 293A cells using recombinantmulti drug resistant HIV-1 (strain MDRC4). Infected cells are treatedwith increasing concentrations of Amprenavir (a), Indinavir (b),Lopinavir (c), Saquinavir (d), REP 2003 (e), REP 2004 (f), REP 2006 (g)and REP 2007 (h). Dose response curves for CNDO (wild type) areindicated in dotted lines and for MDRC4 (drug resistant) are indicatedin solid lines.

FIG. 24. (a) IC50 values from FIGS. 21 and 23 showing fold increases inIC50 values between wild type (CNDO) and drug resistant (MDRC4) strainsof recombinant HIV-1 and (b), plot of fold increases calculated in (a).

FIG. 25. CPE assay conducted in Hep2 cells using RSV (strain A2).Infected cells are treated with increasing concentrations of REP 2004(a), REP 2006 (b), REP 2007 (c) or Ribavirin (d). IC₅₀ values calculatedfrom linear regressions are reported in each graph. Cytotoxicityprofiles in uninfected Hep2 cells are presented for REP 2004 (e), REP2006 (f), REP 2007 (g) or Ribavirin (h).

FIG. 26. Relationship between PS-ODN size and IC₅₀ against RSV. IC₅₀values from FIG. 25 are plotted against the specific size of each PS-ODNtested in FIG. 25 which showed anti-RSV activity.

FIG. 27. CPE assay conducted in LLC-MK2 cells using Coxsackievirus B2(strain Ohio-1). Infected cells are treated with increasingconcentrations of REP 2006 (a). The cytotoxicity profile for REP 2006 isshown in (b).

FIG. 28. A) FP interaction assay showing the ability of PS-ODN randomers(REP 2003, 2004, 2006 and 2007) to compete the interaction of a 20 basePS-ODN randomer bait from FBS. Larger randomers compete moreefficiently. B) and C) Serum protection and improved delivery of REP2006in 293 A cells with DOTAP and Cytofectin. D) and E) Serum protection ofREP 2006 encapsulated with DOTAP or cytofectin measured by FP.

FIG. 29. Determination of viral lysate binding to baits of differentsizes by fluorescence polarization. REP 2032-FL, REP 2003-FL and REP2004-FL were tested for lysate binding in lysates from HSV-1 (a), HIV-1(b) or RSV (c).

FIG. 30. Determination of affinity of PS-ODN randomers for viral lysatesby fluorescence polarization. Using REP 2004-FL as the bait, complexformation with HSV-1 lysate (a), HIV-1 lysate (b) or RSV lysate (c) waschallenged with increasing concentrations of REP 2003, REP 2004, REP2006 or REP 2007.

FIG. 31. REP 2004-FL can bind to HIV-1 p24gag and HIV-1 gp41. Theability of REP 2004-FL to interact with increasing amounts of these twopurified proteins is tested by fluorescence polarization.

FIG. 32. Effect of bait size on p24 and gp41 binding. Baits ofincreasing sizes are tested for their ability to bind to p24gag and gp41by fluorescence polarization.

FIG. 33. The ability of double stranded PS-ODNs to bind to viral lysatesis tested by fluorescence polarization. Single stranded (ss) or doublestranded (ds) phosphorothioated REP 2017 (fluorescently labeled) wasprepared as well as its non-thioated analog (2017U). These baits weretested for binding to HSV-1 and HIV-1 viral lysates.

FIG. 34. The delivery of fluorescently tagged PS-ODNs into cells wasmeasured by incubating 293A cells in the presence of 250 nM REP 2004-FLfor 4 h. Following the incubation, cells were lysed and the relativefluorescence released from the cells upon lysis was measured byfluorometry.

FIG. 35. The ability of 20-mer PS-ODNs of different sequencecompositions to bind to viral lysates is measured by fluorescencepolarization. PS-ODNs 3′ labeled with FITC are incubated in the presenceof 1 ug of HSV-1 (a), HIV-1 (b) or RSV (c) lysates. The binding profilesfor these PS-ODNs is similar in all three viral lysates (see FIG. 35).

FIG. 36. Indirect determination of viral load in infected supernatantsfrom vaccinia infected VERO cells by measuring the CPE induced by thesesupernatants in naive cells. REP 2004, 2006 and 2007 were tested at 10uM while Cidofovir was tested at 50 uM).

FIG. 37. (A) IC50 values generated from a plaque reduction assayconducted in VERO cells using HSV-1 (strain KOS). Infected cells aretreated with increasing concentrations of REP 2006 (N40), REP 2028(G40), REP 2029 (A40), REP 2030 (T40), and REP 2031 (C40) to generateIC50 values. (B) HSV-1 PRA generated IC50 values of the following: N40(REP 2006), AC20 (REP 2055, TC20 (REP 2056), or AG20 (REP 2057).

SELECTED ABBREVIATIONS

-   -   ON: Oligonucleotide    -   ODN: Oligodeoxynucleotide    -   PS: Phosphorothioate    -   PRA: Plaque reduction assay    -   PFU: Plaque forming unit    -   INF A: Influenza A virus    -   HIV: Human immunodeficiency virus (includes both HIV-1 and HIV-2        if not specified)    -   HSV: Herpes simplex virus (includes both HSV-2 and HSV-3 if not        specified)    -   RSV: Respiratory syncytial virus    -   COX: Coxsackievirus    -   DHBV: Duck hepatitis B virus

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is concerned with the identification and use ofantiviral oligonucleotides that act by a non-sequence complementarymechanism, and includes the discovery that for many viruses, theantiviral activity is greater for larger oligonucleotides, and aretypically optimal for oligonucleotides that are 40 nucleotides or morein length.

As described in the Background, a number of antisense oligonucleotides(ONs) have been tested for antiviral activity. However, such antisenseONs are sequence-specific, and typically are about 16-20 nucleotides inlength.

As demonstrated by the results in Examples 1 and 2, the antiviral effectof random PS-ODNs is not sequence specific. Considering the volumes andconcentrations of PS-ODNs used in those tests, it is almosttheoretically impossible that a particular random sequence is present atmore than 1 copy in the mixture. This means than there can be noantisense effect in these PS-ODNs randomers. In the latter example,should the antiviral effect be caused by the sequence-specificity of thePS-ODNs, such effect would thus have to be caused by only one molecule,a result that does not appear possible. For example, for an ON randomer40 bases in length, any particular sequence in the population wouldtheoretically represent only ¼⁴⁰ or 8.27×10⁻²⁵ of the total fraction.Given that 1 mole=6.022×10²³ molecules, and the fact that our largestsynthesis is currently done at the 15 micromole scale, all possiblesequences will not be present and also, each sequence is present mostprobably as only one copy. Of course, one skilled in the art applyingthe teaching of the present invention could also use sequence specificONs, but utilize the non-sequence complementary activity discovered inthe present invention. Accordingly, the present invention is not to berestricted to non-sequence complementary ONs, but disclaims what hasbeen disclosed in the prior art regarding sequence-specific antisenseONs for treating viral infections.

For applicable viruses (including, for example, those for which data isdescribed herein), as the size of the randomer increases, so does itsantiviral potency. It should be pointed out that due to limitations incurrent phosphoramidite-based DNA synthesis, the larger PS-ODNs (e.g.,80- and 120-mers) have a significant contamination of fragments smallerthan the desired size. The weaker effects (on a per base basis) seenwith larger oligos (80 and 120 bp) may reflect the lower concentrationof full-length randomers in these populations and may also reflect adecreased uptake into the cell. It may be possible to achieve muchlarger increases in antiviral activity if larger randomers (>40 bases)of reasonable purity (75% full length) were synthesized or purified,and/or if the cellular uptake of any of these ODNs is facilitated by adelivery system.

In the present invention, randomers (or other oligonucleotides) mayblock viral replication by several mechanisms, including but not limitedto the following: 1. preventing the adsorption or receptor interactionof virions, thus preventing infection, 2. doping the virus assembly orthe packaging of viral genomes into capsids (competing with viral DNA orRNA for packaging), resulting in defective virions, 3. disrupting and orpreventing the formation of capsids during packaging or the interactionof capsid proteins with other structural proteins, resulting ininhibition of viral release or causing the release of defective virions,4. binding to key viral components and preventing or reducing theiractivity, 5. binding to key host components required for viralproliferation.

Without being limited on the mechanism by which the present viralinhibition is achieved, as indicated above there are several possiblemechanisms that could explain and/or predict the inhibitory propertiesof ONs against viral replication. The first of these is that the generalaptameric effect of ONs is allowing for their attachment, either toproteins on the cell surface or to viral proteins, preventing viraladsorption and fusion. The size threshold for effect may be a result ofa certain cumulative charge required for interaction.

A second possible mechanism is that ONs may function within the cell bypreventing packaging and/or assembly of the virus. ONs above a certainsize threshold may compete or interfere with the normal capsid/nucleicacid interaction, preventing the packaging of a functional viral genomeinside new viruses. Alternatively, ONs may prevent the formation of anormal capsid, which could prevent normal viral budding, alter viralstability, or prevent proper virion disassembly upon internalization.

While the mechanism of action is not yet entirely clear, assay resultsdemonstrate that the present ONs can exhibit greater efficacy in viralinhibition compared to the clinical correlates, acyclovir, gancyclovir,Ribavirin, and protease inhibitors. ONs in accordance with the presentinvention could thus be used for treating or preventing viral infection.The viral infections treated could be those caused by human, animal, andplant viruses.

Broad Spectrum Antiviral Activity

According to the conclusions discussed above and the data reportedherein, it appeared that random ONs and ON randomers could havebroad-spectrum antiviral activity with viruses where assembly and/orpackaging and/or encapsidation of the viral genome is a required step inreplication. Therefore to test this hypothesis, several PS-ODN randomersof different sizes were selected to be tested in cellular models ofvarious viral Infections. A number of such tests are described herein inthe Examples, including tests with CMV, HIV-1, RSV, Coxsackie virus B2,DHBV, Hantavirus, Parainfluenza virus, and Vaccinia virus, as well asthe tests on HSV-1 and HSV-2 described in Examples 1 and 2. Despite thehigh activity level exhibited for some of the tested oligonucleotides,an oligo delivery system such as DOTAP, lipofectamine or oligofectaminecould result in much greater efficacies, especially with the larger (≧40bases) randomers.

Conclusions on Broad Spectrum Antiviral Activity

The efficacy studies with different viruses demonstrate that random ONsand randomers display inhibitory properties against a variety ofdifferent viruses. Moreover, these studies support the conclusion thatlarger randomers display greater efficacy for viral inhibition thansmaller randomers. This suggests a common size and/or charge dependentmechanism for the random ONs or ON randomers activity in allencapsidating viruses.

While HSV and CMV are both double-stranded DNA viruses of theherpesviridae family, HIV is a RNA virus from the retroviridae, and RSVa RNA virus from the paramyxoviridae. Given the fact that ON randomerscan inhibit viral function in a variety of different viruses, withoutbeing limited to the mechanisms listed, as discussed above the followingmechanisms are reasonable: A) ONs/ON randomers are inhibiting viralinfection via an aptameric effect, preventing viral fusion with theplasma membrane; and/or B) ONs/ON randomers are preventing or doping theassembly of virions or the packaging of viral DNA within capsidsresulting in defective virions; and/or C)ONs/ON randomers areinterfering with host proteins or components required in the assemblyand/or packaging and/or gene expression of the virus.

Requirement for Antiviral Activity

Since a randomized DNA sequence seems to be sufficient for viralinhibition, it was interesting to see if antiviral activity could bemaintained in the absence of the phosphorothioate modification and alsoif the efficacy was augmented by either choosing a random sequence or aspecific sequence found in the viral genome.

Accordingly, DNA and RNA modifications were investigated with respect totheir effect on the antiviral efficacy of the randomers. Since randomerswork via a non-sequence complementary mechanism, these experiments weredesigned to test the slight changes in nucleic acid conformation andcharge distribution on antiviral efficacy.

To test if ODNs with different nucleotide/nucleoside modifications couldinhibit HSV-1, REP 2024, 2026, 2059, and 2060 were tested in the HSV-1PRA as described in the Examples. REP 2024 (a PS-ODN with a 2-O-Methylmodification to the ribose on 4 bases at both termini of the ODN), REP2026 (a PO-ODN with methylphosphonate modifications to the linkagesbetween the 4 bases at both termini of the ODN), REP 2059 (RNA PS-ODNrandomer 20 bases in length), and REP 2060 (RNA PS-ODN randomer 30 basesin length) showed anti-HSV-1 activity (see FIG. 11).

In the latter example, should the antiviral effect be caused only by theONs consisting of DNA phosphorothioate backbone, such effect would thusbe caused by only one molecule. But other backbones and modificationsgave positive antiviral activity. Of course, one skilled in the artapplying the teaching of the present invention could also use differentchemistry ONs. A modification of the ON, such as, but not limited to, aphosphorothioate modification, appears to be beneficial for antiviralactivity. This is most likely due to the needed charge of ONs and/or therequirement for stabilization of DNA both in the media andintracellularly, and it may also be due to the chirality of the PS-ODNs.

Compound REP 2026 showed an antiviral activity while having a centralportion comprising unmodified PO-nucleotides and 4 methylphosphonatelinkages at both termini protecting from degradation. This indicatesthat PO-ODNs can be used as antivirals while protected from degradation.This protection can be achieved by modifying nucleotides at terminiand/or by using a suitable delivery system as described later.

In general, the sequence composition of the DNA used has little effecton the overall efficacy, whether randomer, random sequence or a specificHSV-1 sequence. However, at intermediate lengths, HSV-1 sequence wasalmost 3× more potent than a random sequence (see FIG. 10). This datasuggests that while specific antisense functionality exists for specificHSV sequences, the non-antisense mechanism (non-sequence complementarymechanism) elucidated herein may represent the predominant part of thisactivity. Indeed, as the ON grows to 40 bases, essentially all of theantiviral activity can be attributed to a non-antisense effect.

Lower Toxicity of Randomer

One goal of using an ON randomer is to lower the toxicity. It is knownthat different sequences may trigger different responses in the animal,such as general toxicity, interaction with serum proteins, andinteraction with immune system (Monteith et al (1998) Toxicol Sci 46:365-375). The mixture of ONs may thus decrease toxic effects because thelevel of any particular sequence will be very low, so that nosignificant interaction due to sequence or nucleotide composition islikely.

Pharmaceutical Compositions

The ONs of the invention may be in the form of a therapeutic compositionor formulation useful for treating (or prophylaxis of) viral diseases,which can be approved by a regulatory agency for use in humans or innon-human animals, and/or against a particular virus or group ofviruses. These ONs may be used as part of a pharmaceutical compositionwhen combined with a physiologically and/or pharmaceutically acceptablecarrier. The characteristics of the carrier may depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain other active factors and/or agents which enhance activity.

Administration of the ONs of the invention used in the pharmaceuticalcomposition or formulation or to practice the method of treating ananimal can be carried out in a variety of conventional ways, such asintraocular, oral ingestion, enterally, inhalation, or cutaneous,subcutaneous, intramuscular, intraperitoneal, intrathecal,intratracheal, or intravenous injection.

The pharmaceutical composition or oligonucleotide formulation of theinvention may further contain other chemotherapeutic drugs for thetreatment of viral diseases, such as, without limitation, Rifampin,Ribavirin, Pleconaryl, Cidofovir, Acyclovir, Pencyclovir, Gancyclovir,Valacyclovir, Famciclovir, Foscarnet, Vidarabine, Amantadine, Zanamivir,Oseltamivir, Resquimod, antiproteases, HIV fusion inhibitors, nucleotideHIV RT inhibitors (e.g., AZT, Lamivudine, Abacavir), non-nucleotide HIVRT inhibitors, Doconosol, Interferons, Butylated Hydroxytoluene (BHT)and Hypericin. Such additional factors and/or agents may be included inthe pharmaceutical composition, for example, to produce a synergisticeffect with the ONs of the invention.

The pharmaceutical composition or oligonucleotide formulation of theinvention may further contain a polymer, such as, without restriction,polyanionic agents, sulfated polysaccharides, heparin, dextran sulfate,pentosan polysulfate, polyvinylalcool sulfate, acemannan,polyhydroxycarboxylates, cellulose sulfate, polymers containingsulfonated benzene or naphthalene rings and naphthalene sulfonatepolymer, acetyl phthaloyl cellulose, poly-L-lysine, sodium caprate,cationic amphiphiles, cholic acid. Polymers are known to affect theentry of virions in cells by, in some cases, binding or adsorbing to thevirion itself. This characteristic of antiviral polymers can be usefulin competing with ONs for the binding, or adsorption to the virion, theresult being an increased intracellular activity of the ONs compared toits extracellular activity.

Exemplary Delivery System

We monitored the uptake of PS-ODN randomers by exposing cultured cellsto fluorescently labeled randomers and then examined the fluorescenceintensity in lysed cells after two rounds of washing. The cellularuptake of cells exposed to 250 nM REP 2004-FL was tested with nodelivery and after encapsulation in one of the following lipid baseddelivery systems; Lipofectamine™ (Invitrogen), Polyfect™ (Qiagen) andOligofectamine™ (Invitrogen). After 4 hours, cells were washed twicewith PBS and lysed using MPER lysis reagent (PROMEGA). FIG. 34 shows therelative fluorescence yield from equivalent numbers of exposed cellswith and without delivery. We observe than in the presence of all threedelivery agents tested, there was a significant increase in theintracellular PS-ODN concentration compared to no delivery.

In keeping with the test results, the use of a delivery system cansignificantly increase the antiviral potency of ON randomers.Additionally, they will serve to protect these compounds from seruminteractions, reducing side effects and maximizing tissue and cellulardistribution.

Although PS-ODNs are more resistant to endogenous nucleases than naturalphosphodiesters, they are not completely stable and are slowly degradedin blood and tissues. A limitation in the clinical application of PSoligonucleotide drugs is their propensity to activate complement on i.v.administration. In general, liposomes and other delivery systems enhancethe therapeutic index of drugs, including ONs, by reducing drugtoxicity, increasing residency time in the plasma, and delivering moreactive drug to disease tissue by extravasation of the carriers throughhyperpermeable vasculature. Moreover in the case of PS-ODN, lipidencapsulation prevents the interaction with potential protein-bindingsites while in circulation (Klimuk et al. (2000) J Pharmacol Exp Ther292: 480-488).

According to our results described herein, an approach is to use adelivery system such as, but without restriction, lipophilic molecules,polar lipids, liposomes, monolayers, bilayers, vesicles, programmablefusogenic vesicles, micelles, cyclodextrins, PEG, iontophoresis, powderinjection, and nanoparticles (such as PIBCA, PIHCA, PHCA, gelatine,PEG-PLA) for the delivery of ONs described herein. The purpose of usingsuch delivery systems are to, among other things, lower the toxicity ofthe active compound in animals and humans, increase cellular delivery,lower the IC50, increase the duration of action from the standpoint ofdrug delivery and protect the oligonucleotides from non-specific bindingwith serum proteins.

We have shown that the antiviral activity of PS-ODN randomers increaseswith increasing size. Moreover this activity is correlated withincreased affinity for viral proteins (in a viral lysate). Since it iswell known in the art that the phosphorothioate modification increasesthe affinity of protein-DNA interaction, we tested the ability ofincreasingly larger PS-ODN randomers to bind to fetal bovine serum (FBS)(FIG. 28 a) using the same FP-based assay used for measuring interactionwith viral lysates. In this assay, 250 ug of non-heat inactivated FBSwas complexed with a fluorescently labeled 20 base PS-ODN randomer,under conditions where the binding (mP value) was saturated. UnlabelledPS-ODN randomers of increasing size (REP 2003, REP 2004, REP 2006 andREP 2007) were used to compete the interaction of FBS with the labeledbait. The results of this test clearly show that as the size of thePS-ODN randomer increases, so does its affinity for FBS. This resultsuggests that the most highly active anti-viral PS-ODNs will also be theones to bind with the highest affinity to proteins.

It is known in the art that one of the main therapeutic problems forphosphorothioate antisense oligonucleotides is their side effects duemainly to this increased interaction with proteins (specifically withserum proteins) as described by Kandimalla and co-workers (Kandimalla etal. (1998) Bioorg. Med. Chem. Lett. 8: 2103-2108). Our data suggestssubstantial benefits by a suitable delivery system capable of deliveringantiviral ONs into the cell while preventing their interaction withserum proteins.

To demonstrate the benefits of a delivery system, we tested twodifferent delivery technologies which are liposomal based; Cytofectinand DOTAP. We measured the delivery of the PS-ODN randomer REP 2006(encapsulated with either Cytofectin or DOTAP) into 293A cells in thepresence of high concentrations of serum (50%) by measuring theintracellular concentration of labeled REP 2006 by fluorometry (FIG. 28b, c). These results show that delivery increases the intracellularconcentration of REP 2006, and also that, in the case of DOTAP, thelevels of intracellular REP 2006 after 24 hours were markedly increased.Finally, we measured the protection of REP2006 from serum proteininteractions by DOTAP (28d) and cytofectin (28e) in our in vitroFP-based interaction assay. Unencapsulated REP 2006 was able to competebound fluorescent oligo from serum but when REP 2006 was encapsulatedwith either DOTAP or cytofectin it was no longer able to compete forserum binding. These data suggest that encapsulation protects oligosfrom serum interaction and will result in a more effective therapeuticeffect with fewer side effects.

Another potential benefit in using a delivery system is to protect theONs from interactions, such as adsorption, with infective virions inorder to prevent amplification of viral infection through differentmechanisms such as increased cellular penetration of virions.

Another approach is to accomplish cell specific delivery by associatingthe delivery system with a molecule(s) that will increase affinity withspecific cells, such molecules being without restriction antibodies,receptor ligands, vitamins, hormones and peptides.

Additional options for delivery systems are provided below.

Linked ODN

In certain embodiments, ONs of the invention are modified in a number ofways without compromising their ability to inhibit viral replication.For example, the ONs are linked or conjugated, at one or more of theirnucleotide residues, to another moiety. Thus, modification of theoligonucleotides of the invention can involve chemically linking to theoligonucleotide one or more moieties or conjugates which enhance theactivity, cellular distribution or cellular uptake, increase transferacross cellular membranes specifically or not, or protecting againstdegradation or excretion, or providing other advantageouscharacteristics. Such advantageous characteristics can, for example,include lower serum interaction, higher viral-protein interaction, theability to be formulated for delivery, a detectable signal, improvedpharmacokinetic properties, and lower toxicity. Such conjugate groupscan be covalently bound to functional groups such as primary orsecondary hydroxyl groups. For example, conjugate moieties can include asteroid molecule, a non-aromatic lipophilic molecule, a peptide,cholesterol, bis-cholesterol, an antibody, PEG, a protein, a watersoluble vitamin, a lipid soluble vitamin, another ON, or any othermolecule improving the activity and/or bioavailability of ONs.

In greater detail, exemplary conjugate groups of the invention caninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, SATE, t-butyl-SATE, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugategroups include cholesterols, lipids, phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, fluorescent nucleobases, and dyes.

Groups that enhance the pharmacodynamic properties, in the context ofthis invention, include groups that improve oligomer cellular uptakeand/or enhance oligomer resistance to degradation and/or protect againstserum interaction. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve oligomeruptake, distribution, metabolism or excretion. Exemplary conjugategroups are described in International Patent Application PCT/US92/09196,filed Oct. 23, 1992, which is incorporated herein by reference in itsentirety.

Conjugate moieties can include but are not limited to lipid moietiessuch as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et at.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et at., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et at.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et at., Nucleosides & Nucleotides, 1995, 14,969-973), oradamantane acetic acid (Manoharan et at., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et at., Biochim. Biophys. Acta,1995, 1264,229-237), or an octadecylamine orhexylaminocarbonyl-oxycholesterol moiety (Crooke et al., J. PharmacolExp. Ther., 1996, 277, 923-937.

The present oligonucleotides may also be conjugated to active drugsubstances, for example without limitation, aspirin, warfarin,phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic.

Exemplary U.S. patents that describe the preparation of exemplaryoligonucleotide conjugates include, for example, U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis incorporated by reference herein in its entirety.

Another approach is to prepare antiviral ONs as lipophilicpro-oligonucleotides by modification with enzymatically cleavable chargeneutralizing adducts such as s-acetylthio-ethyl or s-pivasloylthio-ethyl(Vives et al., 1999, Nucl Acids Res 27: 4071-4076). Such modificationshave been shown to increase the uptake of ONs into cells.

Design of Non-Specific ONs

In another approach, an antiviral ON demonstrating low, preferably thelowest possible, homology with the human (or other subject organism)genome is designed. The goal is to obtain an ON that will show thelowest toxicity due to interactions with human or animal genomesequence(s) and mRNAs. The first step is to produce the desired lengthsequence of the ON, e.g., by aligning nucleotides A, C, G, T in a randomfashion, manually or, more commonly, using a computer program. Thesecond step is to compare the ON sequence with a library of humansequences such as GenBank and/or the Ensemble Human Genome Database. Thesequence generation and comparison can be performed repetitively, ifdesired, to identify a sequence or sequences having a desired lowhomology level with the subject genome. Desirably, the ON sequence is atthe lowest homology possible with the entire genome, while alsopreferably minimizing self interaction.

Non-Specific ONs with Antisense Activity

In another approach, an antiviral non-specific sequence portion(s)is/are coupled with an antisense sequence portion(s) to increase theactivity of the final ON. The non-specific portion of the ONs isdescribed in the present invention. The antisense portion iscomplementary to a viral mRNA.

Non-Specific ONs with a G-Rich Motif Activity

In another approach, an antiviral non-specific sequence portion(s)is/are coupled with a motif portion(s) to improve the activity of thefinal ON. The non-specific portion of the ON is described in the presentinvention. The motif portion can, as non-limiting examples, include,CpG, Gquartet, and/or CG that are described in the literature asstimulators of the immune system. Agrawal et al. (2001) Curr. CancerDrug Targets 3: 197-209.

Non-Watson-Crick ONs

Another approach is to use an ON composed of one type or more ofnon-Watson-Crick nucleotides/nucleosides. Such ONs can mimic PS-ODNswith some of the following characteristics similar to PS-ODNs: a) thetotal charge; b) the space between the units; c) the length of thechain; d) a net dipole with accumulation of negative charge on one side;e) the ability to bind to proteins; f) the ability to bind viralproteins, g) the ability to penetrate cells, h) an acceptabletherapeutic index, i) an antiviral activity. The ON has a preferredphosphorothioate backbone but is not limited to it. Examples ofnon-Watson-Crick nucleotides/nucleosides are described in Kool, 2002,Acc. Chem. Res. 35: 936-943; and Takeshita et al., (1987) J. Biol. Chem.262: 10171-10179 where ODNs containing synthetic abasic sites aredescribed.

Antiviral Polymer

Another approach is to use a polymer mimicking the activity ofphophorothioate ODNs. As described in the literature, several anionicpolymers were shown to have antiviral inhibitory activity. Thesepolymers belong to several classes: (1) sulfate esters ofpolysaccharides (dextrin and dextran sulfates; cellulose sulfate); (2)polymers containing sulfonated benzene or naphthalene rings andnaphthalene sulfonate polymers; (3) polycarboxylates (acrylic acidpolymers); and acetyl phthaloyl cellulose (Neurath et al. (2002) BMCInfect Dis 2: 27); and (4) abasic oligonucleotides (Takeshita et al.,1987, J. Biol. Chem. 262: 10171-10179). Other examples of non-nucleotideantiviral polymers are described in the literature. The polymersdescribed herein mimic PS-ODNs described in this invention and have thefollowing characteristics similar to PS-ODNs: a) the length of thechain; b) a net dipole with accumulation of negative charge on one side;c) the ability to bind to proteins; d) the ability to bind viralprotein, e) an acceptable therapeutic index, f) an antiviral activity.In order to mimic the effect of a PS-ODN, the antiviral polymer maypreferably be a polyanion displaying similar space between its units ascompared to a PS-ODN. It may also have the ability to penetrate cellsalone or in combination with a delivery system.

Antiviral Activity of Double-Stranded PS-ODNs

A random sequence (REP 2017) and its complement (either PS modified orunmodified) are fluorescently labeled as described elsewhere and testedfor their ability to bind to purified HSV-1 and HIV-1 proteins byfluorescence polarization as described in the present invention.Hybridization was verified by acrylamide gel electrophoresis. UnmodifiedREP 2017 (2017U), either single (ss) or double stranded (ds), had nobinding activity in either HSV-1 or HIV-1 lysates. However, PS modifiedREP 2017, either single stranded or double stranded, was capable ofHSV-1 and HIV-1 interaction (see FIG. 33).

According to our results described herein, an approach is to use doublestranded ONs as effective antiviral agents. Preferentially such ONs havea phosphorothioate backbone but may also have other and/or additionalmodifications which increase either their delivery and/or antiviralactivity and/or stability as described herein for single stranded ONs.

In Vitro Assay for Drug Discovery

An in vitro assay is developed based on fluorescence polarization tomeasure the ability of PS-ODNs to bind to viral components, e.g., viralproteins. When a protein (or another interactor) binds to thefluorescently labeled bait, the three dimensional tumbling of the baitin solution is retarded. The retardation of this tumbling is measured byan inherent increase in the polarization of excited light from thelabeled bait. Therefore, increased polarization (reported as adimensionless measure [mP]) is correlated with increased binding.

One methodology is to use as bait a PS-ODN randomer labeled at the 3′end with FITC using an inflexible linker (3′-(6-Fluorescein) CPG). ThisPS-ODN randomer is diluted to 2 nM in assay buffer (10 mM Tris, pH7.2,80 mM NaCl, 10 mM EDTA, 100 mM b-mercaptoethanol and 1% tween 20). Thisoligo is then mixed with an appropriate interactor. In this case, we uselysates of sucrose gradient purified HSV-1 (strain MacIntyre), HIV-1(strain Mn) or RSV (strain A2) suspended in 0.5M KCl and 0.5% TritonX-100 (HSV-1 and HIV-1) or 10 mM Tris, pH7.5, 150 mM NaCl, 1 mM EDTA and0.1% Triton X-100 (RSV). Following bait interaction, the complexes arechallenged with various unlabelled PS-ODNs to assess their ability todisplace the bait from its complex.

In FIG. 29, we show a preliminary test with three baits of differentsizes; 6 (REP 2032-FL), 10 (REP 2003-FL) and 20 bases (REP 2004-FL).These baits were tested for their ability to interact with HSV-1 (FIG.29 a), HIV-1 (FIG. 29 b) and RSV (FIG. 29 c) lysates. In the presence ofany of the viral lysates the degree of binding was dependent on the sizeof the bait used, with 2004-FL displaying the largest shift in mP(binding) in the presence of viral lysate. We note that this is similarto the size dependent antiviral efficacy of PS-ODN randomers. This baitwas then used to assess the ability of PS-ODNs of different sizes tocompete the interaction of the bait with the lysate.

In FIG. 30, the interaction of REP 2004-FL with HSV-1 (FIG. 30 a), HIV-1(FIG. 30 b) and RSV (FIG. 30 c) lysates is challenged with PS-ODNs ofincreasing size. For each viral lysate tested, we note that REP 2003 isunable to compete the bait away from the lysate. The bait interactionwas very strong as revealed by the relatively weak competition elicitedby the REP 2004 (unlabeled bait) competitor. However, it was observedthat as the size of the competitor PS-ODN increased above 20 bases, itsability to displace the bait became more robust. This indicates anincreased affinity to protein components in the viral lysate as thePS-ODN randomer size increases. This phenomenon mirrors the increasedantiviral activity of larger PS-ODN randomers against HSV-1, HSV-2, CMV,HIV-1 and RSV.

The similarity between the efficacy in bait competition and antiviralactivity of PS-ODN randomers indicates that this assay paradigm is agood predictor of antiviral activity. This assay is robust, easy toperform and very stable, making it a very good candidate for a highthroughput screen to identify novel antiviral molecules based not onspecific target identification but on their ability to interact with oneor more components, e.g., viral proteins.

While the exemplary method described herein utilizes fluorescencepolarization to measure interaction with the viral lysate, numeroustechniques are known in the art for monitoring protein interactions, andcan be used in the present methods. These include without restrictionsurface plasmon resonance, fluorescence resonance energy transfer(FRET), enzyme linked immunosorbent assay (ELSIA), gel electrophoresis(to measure mobility shift), isothermal titration and differentialscanning microcalorimetry and column chromatography. These otherdifferent techniques can be applied to measure the interaction of ONswith a viral lysate or component, and thus can be useful in screeningfor compounds which have anti-viral activity.

The method described herein is used to screen for novel compounds fromany desired source, for example, from a library synthesized bycombinatorial chemistry or isolated by purification of naturalsubstances. It can be used to a) determine appropriate size,modifications, and backbones of novel ONs; b) test novel moleculesincluding novel polymers; predict a particular virus' susceptibility tonovel ONs or novel compounds; or d) determine the appropriate suite ofcompounds to maximally inhibit a particular virus.

The increased lysate affinity with larger sized PS-ODN randomerssuggests that the antiviral mechanism of action of PS-ODN randomers isbased on an interaction with one or more viral protein components whichprevents either the infection or correct replication of virions. It alsosuggests that this interaction is charge (size) dependent and notdependent on sequence. As these PS-ODN randomers have a size dependentactivity across multiple viruses spanning several different families, wesuggest that PS-ODN randomers interfere with common, charge dependentprotein-protein interactions, protein-DNA/RNA interactions, and/or othermolecule-molecule interactions. These interactions can include (but arenot limited to):

-   -   a. The interaction between individual capsid subunits during        capsid formation.    -   b. The interaction between the capsid/nucleocapsid protein and        the viral genome.    -   c. The interaction between the capsid and glycoproteins during        budding.    -   d. The interaction between the glycoprotein and its receptor        during infection.    -   e. The interaction between other viral key components involved        in viral replication.

These multiple, simultaneous inhibitions of protein-protein interactionsrepresent a novel mechanism for antiviral inhibition.

Effect of PS-ODN Sequence Composition on Lysate

We monitored the ability of PS-ODNs of different sequences to interactwith several viral lysates. In each case, a 20-mer PS-ODN is labeled atthe 3′ end with FITC as previously described herein. The PS-ODNs testedconsisted of A20, T20, G20, C20, AC10, AG10, TC10, TG10, REP 2004 andREP 2017. Each of these sequences is diluted to 4 nM in assay buffer andincubated in the presence of 1 ug of HSV-1, HIV-1 or RSV lysate.Interaction is measured by fluorescence polarization.

The profile of interaction with all sequences tested is similar in allviral lysates, indicating that the nature of the binding interaction isvery similar. Within each lysate, the PS-ODNs of uniform composition(A20, G20, T20, C20) were the weakest interactors with A20 being theweakest interactor of these by a significant margin. For the rest of thePS-ODNs tested, all of them displayed a similar, strong interaction withthe exception of TG10, which consistently displayed the strongestinteraction in each lysate (see FIG. 35).

Target Identification for PS-ODN Randomers in HIV-I

The ability of PS-ODN randomers to bind to purified HIV-1 proteins istested by fluorescence polarization as described in example 9.Increasing quantities of purified HIV-1 p24 or purified HIV-1 gp41 werereacted with REP 2004-FL (see FIG. 31). We note that for both theseproteins, there is a protein concentration dependent shift influorescence polarization, indicating an interaction with both theseproteins.

The ability of a range of sizes of PS-ODN randomers to bind to theseproteins was also tested using fluorescent versions of REP 2032, REP2003, REP 2004, REP 2006 and REP 2007 (see FIG. 32). We observe that forp24, there is no size dependent interaction with p24 (see FIG. 32 a)however; we did see an increase in gp41 binding in PS-ODN randomerslarger than 20 bases versus those less than 20 bases (see FIG. 32 b).This suggests when PS-ODN randomer length increases above 20 bases,multiple copies of gp41 can bind to individual randomers, increasingtheir polarization.

This is a significant observation as it demonstrates the potential oflarger ONs to sequester structural proteins during viral synthesis andlimit their availability for the formation of new virions.

High Affinity Oligonucleotides

Another approach is a method to enrich or purify antiviral ON(s) havinga higher affinity for viral components, such as viral proteins, than theaverage affinity of the ONs in a starting pool of ONs. The method willthus provide one or more non-sequence complementary ON(s) that willexhibit increased affinity to one or more viral components, e.g., havinga three-dimensional shape contributing to such elevated bindingaffinity. The rationale is that while ON(s) will act as linear moleculesin binding with viral components, they can also fold into a3-dimensional shape that can enhance the interaction with such viralcomponents. Without being limited to the specific technique, highaffinity ONs can be purified or enriched in the following ways.

One method for purifying a high affinity ON, or a plurality of highaffinity ONs, involves using a stationary phase medium with bound viralprotein(s) as an affinity matrix to bind ONs, which can then be elutedunder increasingly stringent conditions (e.g., increasing concentrationof salt or other chaotropic agent, and/or increasing temperture and/orchanges in pH). Such a method can, for example, be carried out by:

-   -   (a) loading a pool of ONs onto an exchange column having a viral        protein or several viral proteins or a viral lysate bound to a        stationary phase;    -   (b) displacing (eluting) bound ONs from the column, e.g., by        using a displacer solution such as an increasing salt solution;    -   (c) collecting fractions of eluted ONs at different salt        concentration;    -   (d) cloning and sequencing eluted ONs from different fractions,        more preferably from a fraction(s) at high salt concentration,        such that the ONs eluted at the high salt concentration have a        greater binding affinity with the viral protein(s); and    -   (e) Testing the activity of sequenced ON(s) in assays such        binding and/or viral inhibiton assay, e.g., a fluorescence        polarization-binding assay as decribed herein and/or in a        cellular viral inhibition assay and/or in an animal viral        inhibition assay.

In a second example, a method derived and modified from the SELEXmethodology (Morris et al (1998) Biochemistry 95: 2902-2907) can be usedfor purifying the high affinity ON. One implementation of such a methodcan be performed as:

-   -   (a) providing a starting ON pool material, for example, a        collection of synthetic random ONs containing a high number of        sequences, e.g., one hundred trillion (10¹⁴) to ten quadrillion        (10¹⁶) different sequences. Each ON molecule contains a segment        of random sequence flanked by primer-binding sequences at each        end to facilitate polymerase chain reaction (PCR). Because the        nucleotide sequences of essentially all of the molecules are        unique, an enormous number of structures are sampled in the        population. These structures determine each molecule's        biochemical properties, such as the ability to bind a given        viral target molecule;    -   (b) contacting ONs with a viral protein or several viral        proteins or a viral lysate;    -   (c) selecting ONs that bind to viral protein(s), using a        partition technique(s) that can partition bound and unbound ONs,        such as native gel shifts and nitrocellulose filtration. Either        of these methods physically separates the bound species from the        unbound species, allowing preferential recovery of those        sequences that bind best. Also, to select ON (s) that bind to a        small protein, it is desirable to attach the target to a solid        support and use that support as an affinity purification matrix.        Those molecules that are not bound get washed off and the bound        ones are eluted with free target, again physically separating        bound and unbound species;    -   (d) amplifying the eluted binding ON(s), e.g., by using PCR        using primers hybridizing with both flanking sequences of ONs;    -   (e) steps (b) (c) and (d) can be performed multiple times (i.e.,        multiple cycles or rounds of enrichment and amplification) in        order to preferentially recover ONs that display the highest        binding affinity to viral protein(s). After several cycles of        enrichment and amplification, the population is dominated by        sequences that display the desired biochemical property;    -   (f) cloning and sequencing one or more ONs selected from from an        enrichment cycle, e.g., the last such cycle; and    -   (g) testing the binding and/or activity of sequenced ON(s) in        assays, e.g., in a fluorescence polarization binding assay as        decribed herein and/or in a cellular viral inhibition assay        and/or in an animal viral inhibition assay.

Another approach is to apply a modification of a split synthesismethodology to create one-bead one-PS-ODN and one-bead one-PS2-ODNlibraries as described in Yang et al (2002) Nucl. Acids Res. 30(e132):1-8. Binding and selection of specific beads to viral proteins can bedone. Sequencing both the nucleic acid bases and the positions of anythioate/dithioate linkages can be carried out by using a PCR-basedidentification tag of the selected beads. This approach can allow forthe rapid and convenient identification of PS-ODNs or PS2-ODNs that bindto viral proteins and that exhibit potent antiviral properties.

Once the specific sequences that bind to the viral proteins with highaffinity are determined (e.g., by amplification and sequencing ofindividual sequences), one or more such high affinity sequences can beselected and synthesized (e.g., by either chemical or enzymaticsynthesis) to provide a preparation of high affinity ON(s), which can bemodified to improve their activity, including improving theirpharmacokinetic properties. Such high affinity ONs can be used in thepresent invention.

Prion Diseases

Another approach is used in an alternative embodiment of the presentinvention for the treatment, the control of the progression, or theprevention of prion disease. This fatal neurodegenerative disease isinfectious and can affect both humans and animals. Structural changes inthe cellular prion protein, PrPC to its scrapie isoform, PrPSC, areconsidered to be the obligatory step in the occurrence and propagationof the prion disease. Amyloid polymers are associated withneuropathology of the prion disease.

The incubation of a prion protein fragment and double stranded nucleicacid results in the formation of amyloid fibres (Nandi et al (2002), JMol Biol 322: 153-161). ONs having affinity to proteins such asphosphorothioates are used to compete or inhibit the interaction ofdouble stranded nucleic acid with the PrPC and consequently stop theformation of the amyloid polymers. Such ONs of different sizes anddifferent compositions can be used in an appropriate delivery form totreat patients suffering from prion diseases or for prophylaxis in highrisk situations. Such interfering ONs can be identified by measuringfolding changes of amyloid polymerase as described by Nandi et al.(supra) in the presence of test ONs.

Putative Viral Etiologies

Another approach is used in another embodiment of the present inventionfor the treatment or prevention of diseases or conditions with putativeviral etiologies as described without limitation in the followingexamples. Viruses are putative causal agents in diseases and conditionsthat are not related to a primary viral infection. For example,arthritis is associated with HCV (Olivieri et al. (2003) Rheum Dis ClinNorth Am 29: 111-122), Parvovirus B19, HIV, HSV, CMV, EBV, and VZV(Stahl et al. (2000) Clin Rheumatol 19: 281-286). Other viruses havealso been identified as playing a role in different diseases. Forexample, influenza A in Parkinson's disease (Takahashi et al. (1999),Jpn J Infect Dis 52: 89-98), Coronavirus, EBV and other viruses inMultiple Sclerosis (Talbot et al (2001) Curr Top Microbiol Immunol 253:247-71); EBV, CMV and HSV-6 in Chronic Fatigue Syndrome (Lerner et al.(2002) Drugs Today 38: 549-561); and paramyxoviruses in asthma (Walteret al (2002) J Clin Invest 110: 165-175) and in Paget's disease; andHBV, HSV, and influrenza in Guillain-Barre Syndrome.

Because of these etiologies, inhibition of the relevant virus using thepresent invention can delay, slow, or prevent development of thecorresponding disease or condition, or at least some symptoms of thatdisease.

Oligonucleotide Modifications and Synthesis

As indicated in the Summary above, modified oligonucleotides are usefulin this invention. Such modified oligonucleotides include, for example,oligonucleotides containing modified backbones or non-naturalinternucleoside linkages. Oligonucleotides having modified backbonesinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone.

Such modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, carboranyl phosphate andborano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity typically include a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Preparation of oligonucleotides with phosphorus-containing linkages asindicated above are described, for example, in U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of whichis incorporated by reference herein in its entirety.

Some exemplary modified oligonucleotide backbones that do not include aphosphorus atom have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts. Particularlyadvantageous are backbone linkages that include one or more chargedmoieties. Examples of U.S. patents describing the preparation of thepreceding oligonucleotides include U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437;5,792,608; 5,646,269 and 5,677,439, each of which is incorporated byreference herein in its entirety.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. For example, such oligonucleotides can include one of thefollowing 2′-modifications: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C, to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl, or 2′-O—(O-carboran-1-yl)methyl.Particular examples are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)˜OCH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to 10. Other exemplaryoligonucleotides include one of the following 2′-modifications: C₁ toC₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃. OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide. Examples include 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxygroup; 2′-dimethy-laminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, alsoknown as 2′-DMAOE; and 2′-dimethylaminoethoxyethoxy (also known as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include Locked Nucleic Acids (LNAs) in which the2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugarring thereby forming a bicyclic sugar moiety. The linkage can be amethelyne (—CH₂—)— group bridging the 2′ oxygen atom and the 4′ carbonatom wherein n is 1 or 2. LNAs and preparation thereof are described inWO 98/39352 and WO 99/14226, which are incorporated herein by referencein their entireties.

Other modifications include sulfur-nitrogen bridge modifications, suchas locked nucleic acid as described in Orum et al. (2001) Curr. Opin.Mol. Ther. 3: 239-243.

Other modifications include 2′-methoxy(2′-O—CH₃),2′-methoxyethyl(2′O—CH₂—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂),2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro(2′-F). The 2′-modification may be in the arabino (up) position or ribo(down) position. Similar modifications may also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotidesand the 5′ position of the 5′ terminal nucleotide. Oligonucleotides mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Exemplary U.S. patents describing the preparationof such modified sugar structures include, for example, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393, 878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567, 811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; 5,792, 747; and 5,700,920, each of which is incorporated byreference herein in its entirety.

Still other modifications include an ON concatemer consisting ofmultiple oligonucleotide sequences joined by a linker(s). The linkermay, for example, consist of modified nucleotides or non-nucleotideunits. In some embodiments, the linker provides flexibility to the ONconcatemer. Use of such ON concatemers can provide a facile method tosynthesize a final molecule, by joining smaller of igonucleotidebuilding blocks to obtain the desired length. For example, a 12 carbonlinker (C12 phosphoramidite) can be used to join two or more ONconcatemers and provide length, stability, and flexibility.

As used herein, “unmodified” or “natural” bases (nucleobases) includethe purine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U). Oligonucleotides may alsoinclude base modifications or substitutions. Modified bases includeother synthetic and naturally-occurring bases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl(—C═C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additionalmodified bases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine(2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases mayalso include those in which the purine or pyrimidine base is replacedwith other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone. Further nucleobases include thosedescribed in U.S. Pat. No. 3,687,808, those disclosed in The ConciseEncyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993.

Another modification includes phosphorodithioate linkages. Knowing thatphosphorodithioate ODNs (PS2-ODNs) and PS-ODNs have a similar bindingaffinity to proteins (Tonkinson et al. (1994) Antisense Res. Dev. 4: 269-278)(Cheng et al. (1997) J. Mol. Recogn. 10: 101-107) and knowing thata possible mechanism of action of ODNs is binding to viral proteins, itcould be desirable to include phosphorodithioate linkages on theantiviral ODNs described in this invention.

Another approach to modify ODNs is to produce stereodefined orstereo-enriched ODNs as described in Yu at al (2000) Bioorg. Med. Chem.8: 275-284 and in Inagawa et al. (2002) FEBS Lett. 25: 48-52. ODNsprepared by conventional methods consist of a mixture of diastereomersby virtue of the asymmetry around the phosphorus atom involved in theinternucleotide linkage. This may affect the stability of the bindingbetween ODNs and viral components such as viral proteins. Previous datashowed that protein binding is significantly stereo-dependent (Yu etal.). Thus, using stereodefined or stereo-enriched ODNs could improvetheir protein binding properties and improve their antiviral efficacy.

The incorporation of modifications such as those described above can beutilized in many different incorporation patterns and levels. That is, aparticular modification need not be included at each nucleotide orlinkage in an oligonucleotide, and different modifications can beutilized in combination in a single oligonucleotide, or even in a singlenucleotide.

Oligonucleotide Synthesis

The present oligonucleotides can by synthesized using methods known inthe art. For example, unsubstituted and substituted phosphodiester (P═O)oligonucleotides can be synthesized on an automated DNA synthesizer(e.g., Applied Biosystems model 380B) using standard phosphoramiditechemistry with oxidation by iodine. Phosphorothioates (P═S) can besynthesized as for the phosphodiester oligonucleotides except thestandard oxidation bottle can be replaced by 0.2 M solution of311-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for thestep-wise thioation of the phosphite linkages. The thioation wait stepcan be increased to 68 sec, followed by the capping step. After cleavagefrom the CPG column and deblocking in concentrated ammonium hydroxide at55° C. (18 h), the oligonucleotides can be purified by precipitatingtwice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.

Phosphinate oligonucleotides can be prepared as described in U.S. Pat.No. 5,508,270; alkyl phosphonate oligonucleotides can be prepared asdescribed in U.S. Pat. No. 4,469,863; 3′-Deoxy-3′-methylene phosphonateoligonucleotides can be prepared as described in U.S. Pat. Nos.5,610,289 and 5,625,050; phosphoramidite oligonucleotides can beprepared as described in U.S. Pat. No. 5,256,775 and U.S. Pat. No.5,366,878; alkylphosphonothioate oligonucleotides can be prepared asdescribed in published PCT applications PCT/US94/00902 andPCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively);3′-Deoxy-3′-amino phosphoramidate oligonucleotides can be prepared asdescribed in U.S. Pat. No. 5,476,925; Phosphotriester oligonucleotidescan be prepared as described in U.S. Pat. No. 5,023,243; boranophosphate oligonucleotides can be prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198; methylenemethylimino linkedoligonucleotides, also identified as MMI linked oligonucleotides,methylenedimethyl-hydrazo linked oligonucleotides, also identified asMDII linked oligonucleotides, and methylenecarbonylamino linkedoligonucleotides, also identified as amide-3 linked oligonucleotides,and methyleneaminocarbonyl linked oligo-nucleotides, also identified asamide-4 linked oligonucleo-sides, as well as mixed backbone compoundshaving, for instance, alternating MMI and P═O or P═S linkages can beprepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677,5,602,240 and 5,610,289; formacetal and thioformacetal linkedoligonucleotides can be prepared as described in U.S. Pat. Nos.5,264,562 and 5,264,564; and ethylene oxide linked oligonucleotides canbe prepared as described in U.S. Pat. No. 5,223,618. Each of the citedpatents and patent applications is incorporated by reference herein inits entirety.

Oligonucleotide Formulations and Pharmaceutical Compositions

The present oligonucleotides can be prepared in an oligonucleotideformulation or pharmaceutical composition. Thus, the presentoligonucleotides may also be admixed, encapsulated, conjugated orotherwise associated with other molecules, molecule structures ormixtures of compounds, as for example, liposomes, receptor targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption. Exemplary United States patentsthat describe the preparation of such uptake, distribution and/orabsorption assisting formulations include, for example, U.S. Pat. Nos.5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158;5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556;5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619;5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528;5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of whichis incorporated herein by reference in its entirety.

The oligonucleotides, formulations, and compositions of the inventioninclude any pharmaceutically acceptable salts, esters, or salts of suchesters, or any other compound which, upon administration to an animalincluding a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to prodrugs and pharmaceuticallyacceptable salts of the compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular embodiments, prodrug versionsof the present oligonucleotides are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in Gosselin et al., WO 93/24510 and in Imbach et al., WO94/26764 and U.S. Pat. No. 5,770,713, which are hereby incorporated byreference in their entireties.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the present compounds: i.e.,salts that retain the desired biological activity of the parent compoundand do not impart undesired toxicological effects thereto. Many suchpharmaceutically acceptable salts are known and can be used in thepresent invention.

For oligonucleotides, useful examples of pharmaceutically acceptablesalts include but are not limited to salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; acid addition salts formed with inorganicacids, for example hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and salts formed fromelemental anions such as chlorine, bromine, and iodine.

The present invention also includes pharmaceutical compositions andformulations which contain the antiviral oligonucleotides of theinvention. Such pharmaceutical compositions may be administered in anumber of ways depending upon whether local or systemic treatment isdesired and upon the area to be treated. For example, administration maybe topical (including ophthalmic and to mucous membranes includingvaginal and rectal delivery); pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal; intranasal; epidermal and transdermal; oral; orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, laurie acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Exemplary surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Exemplary bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Exemplaryfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further exemplary penetrationenhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may bedelivered orally in granular form including sprayed dried particles, orcomplexed to form micro or nanoparticles. Oligonucleotide complexingagents include poly-amino acids; polyimines; polyacrytates;polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationizedgelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) andstarches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,pollulans, celluloses, and starches. Particularly advantageouscomplexing agents include chitosan, N-trimethytchitosan, poly-L-lysine,polyhistidine, polyorithine, polyspermines, protamine,polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE),polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate),poly(ethylcyanoacrylate), poly(butylcyanoacrylatc),poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate),DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin andDEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lacticacid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, andpolyethyleneglycol (PEG).

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shakingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The formulations and compositions of the present invention may beprepared and formulated as emulsions. Emulsions are typicallyheterogenous systems of one liquid dispersed in another in the form ofdroplets usually exceeding 0.1 μm in diameter. (Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (lids.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et at., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: non-ionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong inter-facial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid, Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailabiity standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically micro-emulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML31O), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschet, Met/i. Find.Exp. Clin. Pharmacol., 1993, 13, 205). Micro-emulsions afford advantagesof improved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset at., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Set, 1996, 85,138-143). Often microemulsions may form spontaneously whentheir components are brought together at ambient temperature. This maybe particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucteotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles offerspecificity and extended duration of action for drug delivery. Thus, asused herein, the term “liposome” refers to a vesicle composed ofamphiphilic lipids arranged in a spherical bilayer or bilayers, i.e.,liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion typically contains the composition to be delivered. Inorder to cross intact mammalian skin, lipid vesicles must pass through aseries of fine pores, each with a diameter less than 50 nm, under theinfluence of a suitable transdermal gradient. Therefore, it is desirableto use a liposome which is highly deformable and able to pass throughsuch fine pores. Additional factors for liposomes include the lipidsurface charge, and the aqueous volume of the liposomes.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245).

For topical administration, there is evidence that liposomes presentseveral advantages over other formulations. Such advantages includereduced side-effects related to high systemic absorption of theadministered drug, increased accumulation of the administered drug atthe desired target, and the ability to administer a wide variety ofdrugs, both hydrophilic and hydrophobic, into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin, generally resulting intargeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etat., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs. TheDNA is thus entrapped in the aqueous interior of these liposomes.pH-sensitive liposomes have been used, for example, to deliver DNAencoding the thymidine kinase gene to cell monolayers in culture (Zhouet al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et at., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18,259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasone™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et at. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome include one ormore glycolipids, such as monosialoganglioside G_(M1), or is derivatizedwith one or more hydrophilic polymers, such as a polyethylene glycol(PEG) moiety. Without being bound by any particular theory, it isbelieved that for sterically stabilized liposomes containinggangliosides, sphingomyelin, or PEG-derivatized lipids, the increase incirculation half-life of these sterically stabilized liposomes is due toa reduced uptake into cells of the reticuloendothelial system (RES)(Allen et at., FEBS Lett., 1987, 223, 42; Wu et al., Cancer Research,1993, 53, 3765).

Various liposomes that include one or more glycolipids have beenreported in Papahadjopoulos et al., Ann. N.Y. Acad. Sci., 1987, 507, 64(monosiatoganglioside G_(M1), galactocerebroside sulfate andphosphatidylinositol); Gabizon et at., Proc. Natl. Acad. Sci. USA.,1988, 85, 6949; Allen et al., U.S. Pat. No. 4,837,028 and InternationalApplication Publication WO 88/04924 (sphingomyelin and the gangliosideG_(M1) or a galactocerebroside sulfate ester); Webb et al., U.S. Pat.No. 5,543,152 (sphingomyelin); Lim et al., WO 97/13499(1,2-sn-dimyristoylphosphatidylcholine).

Liposomes that include lipids derivatized with one or more hydrophilicpolymers, and methods of preparation are described, for example, inSunamoto et al., Bull. Chem. Soc. Jpn., 1980, 53, 2778 (a nonionicdetergent, 2C₁₂15G, that contains a PEG moiety); Illum et al., FEBSLett., 1984, 167, 79 (hydrophilic coating of polystyrene particles withpolymeric glycols); Sears, U.S. Pat. Nos. 4,426,330 and 4,534,899(synthetic phospholipids modified by the attachment of carboxylic groupsof polyalkylene glycols (e.g., PEG)); Klibanov et al., FEBS Left., 1990,268, 235 (phosphatidylethanolamine (PE) derivatized with PEG or PEGstearate); Blume et al., Biochimica et Biophysica Acta, 1990, 1029, 91(PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from thecombination of distearoylphosphatidylethanolamine (DSPE) and PEG);Fisher, European Patent No. EP 0 445 131 B1 and WO 90/04384 (covalentlybound PEG moieties on liposome external surface); Woodle et al., U.S.Pat. Nos. 5,013,556 and 5,356,633, and Martin et al., U.S. Pat. No.5,213,804 and European Patent No. EP 0 496 813 B1 (liposome compositionscontaining 1-20 mole percent of PE derivatized with PEG); Martin et al.,WO 91/05545 and U.S. Pat. No. 5,225,212 and in Zalipsky et al., WO94/20073 (liposomes containing a number of other lipid-polymerconjugates); Choi et al., WO 96/10391 (liposomes that includePEG-modified ceramide lipids); Miyazaki et al., U.S. Pat. No. 5,540,935,and Tagawa et al., U.S. Pat. No. 5,556,948 (PEG-containing liposomesthat can be further derivatized with functional moieties on theirsurfaces).

Liposomes that include nucleic acids have been described, for example,in Thierry et al., WO 96/40062 (methods for encapsulating high molecularweight nucleic acids in liposomes); Tagawa et al., U.S. Pat. No.5,264,221 (protein-bonded liposomes containing RNA); Rahman et al., U.S.Pat. No. 5,665,710 (methods of encapsulating oligodeoxynucleotides inliposomes); Love et al., WO 97/04787 (liposomes that include antisenseoligonucleotides).

Another type of liposome, transfersomes are highly deformable lipidaggregates which are attractive for drug delivery vehicles. (Cevc etal., 1998, Biochim Biophys Acta. 1368(2): 201-15.) Transfersomes may bedescribed as lipid droplets which are so highly deformable that they canpenetrate through pores which are smaller than the droplet.Transfersomes are adaptable to the environment in which they are used,for example, they are shape adaptive, self-repairing, frequently reachtheir targets without fragmenting, and often self-loading. Transfersomescan be made, for example, by adding surface edge-activators, usuallysurfactants, to a standard liposomal composition.

Surfactants

Surfactants are widely used in formulations such as emulsions (includingmicroemulsions) and liposomes. The most common way of classifying andranking the properties of the many different types of surfactants, bothnatural and synthetic, is by the use of the hydrophile/lipophile balance(HLB). The nature of the hydrophilic group (also known as the “head”)provides the most useful means for categorizing the differentsurfactants used in formulations (Rieger, in Pharmaceutical DosageForms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants are widely used inpharmaceutical and cosmetic products and are usable over a wide range ofpH values, and with typical HLB values from 2 to about 18 depending onstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters; andnonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost commonly used members of the nonionic surfactant class.

Surfactant molecules that carry a negative charge when dissolved ordispersed in water are classified as anionic. Anionic surfactantsinclude carboxylates such as soaps, acyl lactylates, acyl amides ofamino acids, esters of sulfuric acid such as alkyl sulfates andethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates,acyl isothionates, acyl laurates and sulfosuccinates, and phosphates.The alkyl sulfates and soaps are the most commonly used anionicsurfactants.

Surfactant molecules that carry a positive charge when dissolved ordispersed in water are classified as cationic. Cationic surfactantsinclude quaternary ammonium salts and ethoxylated amines, with thequaternary ammonium salts used most often.

Surfactant molecules that can carry either a positive or negative chargeare classified as amphoteric. Amphoteric surfactants include acrylicacid derivatives, substituted alkylamides, N-alkylbetaines andphosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed in Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In some embodiments, penetration enhancers are used in or with acomposition to increase the delivery of nucleic acids, particularlyoligonucleotides, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating nonsurfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classesof penetration enhancers is described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. These penetration enhancers include, for example, sodiumlauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether) (Lee et at., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252), each of which is incorporated herein by reference in itsentirety.

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and diglycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654),each of which is incorporated herein by reference in its entirety.

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(U DCA), sodium tauro-24,25-dihydro-fusidate (STDH F), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: In the present context, chelating agents can beregarded as compounds that remove metallic ions from solution by formingcomplexes therewith, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Without limitation, chelating agents include disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990,14,43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds are compounds that do notdemonstrate significant chelating agent or surfactant activity, butstill enhance absorption of oligonucleotides through the alimentarymucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33). Examples of such penetration enhancers includeunsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and nonsteroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions andformulations of the present invention. For example, cationic lipids,such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(Lollo et al., PCT Application WO 97/30731), are also known to enhancethe cellular uptake of oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, often with an excess of the latter substance, can result in asubstantial reduction of the amount of nucleic acid recovered in theliver, kidney or other extracirculatory reservoirs. For example, therecovery of a partially phosphorothioate oligonucleotide in hepatictissue can be reduced when it is coadministered with polyinosinic acid,dextran sulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995,5,115-121; Takakura et al., Antisense & Nucl.Acid Drug Dev., 1996, 6, 177-183), each of which is incorporated hereinby reference in its entirety.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal, and is typically liquid or solid. Apharmaceutical carrier is generally selected to provide for the desiredbulk, consistency, etc., when combined with a nucleic acid and the othercomponents of a given pharmaceutical composition, in view of theintended administration mode. Typical pharmaceutical carriers include,but are not limited to, binding agents (e.g., pregelatinized maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.);fillers (e.g., lactose and other sugars, microcrystalline cellulose,pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates orcalcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate,talc, silica, colloidal silicon dioxide, stearic acid, metallicstearates, hydrogenated vegetable oils, corn starch, polyethyleneglycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g.,starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodiumlauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Other Pharmaceutical Composition Components

The present compositions may additionally contain other componentsconventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the compositions of thepresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of thepresent invention. The formulations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings and/or aromatic substances andthe like which do not deleteriously interact with the nucleic acid(s) ofthe formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran, and/or stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antiviral oligonucleotides and (b) one ormore other chemotherapeutic agents which function by a differentmechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethytmetamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), coichicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-EU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toRibavirin, cidofovir, vidarabine, acyclovir and ganciclovir, may also becombined in compositions of the invention. See, generally, The MerckManual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987,Rahway, N.J., pages 2499-2506 and 46-49, respectively). Othernon-oligonucleotide chemotherapeutic agents are also within the scope ofthis invention. Two or more combined compounds may be used together orsequentially.

EXAMPLES Example 1 Herpes Simplex Virus

Herpes simplex virus (HSV) affects a significant proportion of the humanpopulation. It was found in the present invention that random ODNs orODN randomers inhibited the infectivity of viruses such as HSV. Usingcellular HSV replication assays in VERO cells (susceptible to HSV-1(strain KOS) and HSV-2 (strain MS2) infection) it was found that asingle stranded PS-ODN complementary to the HSV origin of replicationinhibited replication of HSV-1 and HSV-2. Surprisingly, control PS-ODNscomplementary to human (343 ARS) and plasmid (pBR322/pUC) origins alsoinhibited viral infectivity. Experiments with random sequence PS-ODNsand PS-ODN randomers demonstrated that inhibition of viral infectionincreased with increasing ODN size. These data show that ONs are potentantiviral agents useful for therapeutic treatment of viral infection.

The inventors have theorized that a potential mechanism for blocking thespread of viruses such as HHVs was to prevent the replication of itsDNA. With this in mind, phosphorothioate oligonucleotides (ODNs)complementary to the origin of replication of HSV1 and HSV2 wereintroduced into infected cells. These ODNs would cause DNA triplexformation at the viral origin of replication, blocking the associationof necessary trans-acting factors and viral DNA replication. Surprisingresults are presented herein of these experiments which show that, in anexperimental paradigm, the potency of ODNs in inhibiting viral infectionincreases as their size (length) increases.

Inhibition of HSV-1

The ability of PS-ODNs to inhibit HSV-1 is measured in a plaquereduction assay (PRA). Immortalized African Green Monkey kidney (VERO)cells are cultured at 37° C. and 5% CO₂ in MEM (minimal essentialmedium) plus 10% fetal calf serum supplemented with gentamycin,vancomycin and amphoterecin B. Cells are seeded in 12 well plates at adensity which yields a confluent monolayer of cells after 4 days ofgrowth. Upon reaching confluency, the media is changed to contain only5% serum plus supplements as described above and cells are then exposedto HSV-1 (strain KOS, approximately 40-60 PFU total) in the presence ofthe test compound for 90 minutes. After viral exposure, the media isreplaced with new “overlay” media containing 5% serum, 1% humanimmunoglobulins, supplements as described above and the test compound.Plaque counting is performed 3-4 days post infection following formalinfixation and cresyl violet staining of infected cultures.

All ONs (except where noted otherwise) were synthesized at theUniversity of Calgary Core DNA Services lab. ONs (see table 1) areprepared on a 1 or 15 micromol synthesis scale, deprotected and desaltedon a 50 cm Sephadex G-25 column. The resulting ONs are analyzed by UVshadowing gel electrophoresis and are determined to contain ˜95% of thefull length, n-1 and n-2 oligo and up to 5% of shorter oligo species(these are assumed to have random deletions). For random oligosynthesis, adenine, guanosine, cytosine and thymidine amidites are mixedtogether in equimolar quantities to maximize the randomness ofincorporation at each position of the ODNs during synthesis.

To test if PS-ODNs could inhibit HSV-1, REP 1001, 2001 and 3007 aretested in the HSV-1 PRA. It is expected that only REP 2001 will show anyactivity as this PS-ODN is directed against the origin of replication inHSV (the other two are directed against replication origins in humansand plasmids). However all three PS-ODNs showed anti-HSV-1 activity (seeFIG. 1). Moreover, the potentcy of the anti-HSV-1 effect is dependent onthe size of the oligo (see FIG. 2).

To confirm the size dependence and relative sequence independence ofPS-ODNs on anti-HSV-1 activity, we tested PS-ODNs that vary in size (REP2002, 2003, 2004, 2005 and 2006). These PS-ODNs are rendered inert withrespect to sequence specific effects by synthesizing each base as a“wobble” (N) so that each PS-ODN actually represents a population ofdifferent random sequences with the same size, these PS-ODNs are termed“randomers”. When these oligos are tested in the HSV-1 PRA, we find thatoligos 10 bases or lower have no detectable anti-HSV-1 activity but asthe size of the PS-ODN increases above 10 bases, the potency alsoincreases (IC₅₀ decreases, see FIGS. 3 and 4). We also note that PS-ODNsgreater than 20 bases had IC₅₀ values significantly lower than aclinically accepted anti-HSV-1 drug, acyclovir (see FIG. 4).

To better define the effective size range for PS-ODN anti-HSV-1activity, we tested PS-ODN randomers covering a broader range of sizesfrom 10 to 120 bases (see FIGS. 5 and 6). We discovered that oligos 12bases and larger have detectable anti-HSV-1 activity and that theefficacy against HSV-1 also increases with increased PS-ODN randomerlength at least up to 120 bases. However, the increases in efficacy perbase increase in size are smaller in PS-ODN randomers greater than 40bases (see FIG. 7).

To compare the efficacy of non-PS-ODN randomers, a random sequencePS-ODN and a HSV-1 specific sequence PS-ODN, we tested these three typesof modifications in ODNs 10, 20 and 40 bases in size (see FIGS. 8 and9). Unmodified ODN randomers have no detectable anti-HSV-1 activity attested sizes (see FIG. 8 a-c). Both random sequence and specific HSV-1sequence PS-ODNs show size dependent anti-HSV-1 activity (no activity isobserved at 10 bases for either of these modifications, see FIGS. 8 dand g). A comparison of random sequence, specific HSV-1 sequence andrandomer PS-ODNs (see FIG. 10) shows that for PS-ODNs 20 bases inlength, there is an enhancement of anti-HSV-1 activity with the specificHSV-1 sequence but that at 40 bases in length, all modifications,whether randomer, random sequence or specific HSV-1 sequence wereequally efficacious against HSV-1.

To the best of our knowledge, this is the first time IC50s for HSV-1 aslow as 0.059 μM and 0.043 μM are reported for PS-ODNs.

Example 2 Inhibition of HSV-2

The ability of PS-ODNs to inhibit HSV-2 is measured by PRA. ImmortalizedAfrican Green Monkey kidney (VERO) cells are cultured at 37° C. and 5%CO₂ in MEM plus 10% fetal calf serum supplemented with gentamycin,vancomycin and amphoterecin B. Cells are seeded in 12 well plates at adensity which yields a confluent monolayer of cells after 4 days ofgrowth. Upon reaching confluency, the media is changed to contain only5% serum plus supplements as described above and cells are then exposedto HSV-2 (strain MS2, approximately 40-60 PFU total) in the presence ofthe test compound for 90 minutes. After viral exposure, the media isreplaced with new “overlay” media containing 5% serum, 1% humanimmunoglobulins, supplements as described above and the test compound.Plaque counting is performed 3-4 days post infection following formalinfixation and cresyl violet staining of infected cultures.

To test if PS-ODNs could inhibit HSV-2, REP 1001, 2001 and 3007 aretested in the HSV-2 PRA. It is expected that only REP 2001 will show anyactivity as this PS-ODN is directed against the origin of replication inHSV-1/2 (the other two are directed against replication origins inhumans and plasmids), however all three PS-ODNs showed anti-HSV-2activity (see FIG. 12). Moreover, the potency of the anti-HSV-2 effectis dependent on the size of the PS-ODN and independent of the sequence(see FIG. 13).

To confirm the size dependence and sequence independence of PS-ODNs onanti-HSV-2 activity, we test PS-ODNs that vary in size (REP 2001, 2002,2003, 2004, 2005 and 2006). These PS-ODNs are rendered inert withrespect to sequence specific effects by synthesizing each base as a“wobble” (N) so that each PS-ODN actually represents a population ofdifferent random sequences with the same size, these PS-ODNs are termed“randomers”. When these PS-ODNs are tested in the HSV-2 PRA, we findthat PS-ODNs 10 bases or lower had no detectable anti-HSV-2 activity butas the size of the PS-ODN increases above 10 bases, the potency alsoincreases (IC₅₀ decreases, see FIGS. 14 and 15). We also noted thatPS-ODNs greater than 20 bases had IC₅₀ values significantly lower than aclinically accepted anti-HSV-2 drug, acyclovir™ (see FIG. 15).

To the best of our knowledge, this is the first time an IC50 for HSV-2as low as 0.012 μM has been reported for a PS-ODN.

To determine if non-specific sequence composition has an effect on ONantiviral activity, several PS-ODNs of equivalent size but differing intheir sequence composition were tested for anti-HSV1 activity in theHSV-1 PRA. The PS-ODNs tested were REP 2006 (N20), REP 2028 (G40), REP2029 (A40), REP 2030 (T40) and REP 2031 (C40). The IC50 values generatedfrom the HSV-1 PRA (see FIG. 37) show that REP 2006 (N40) was clearlythe most active of all sequences tested while REP 2029 (A40) was theleast active. We also note that, all the other PS-ODNs weresignificantly less active than N40 with their rank in terms of efficacybeing N40>C40>T40>A40>>G40.

We also tested the efficacy of different PS ODNs having varying sequencecomposition with two different nucleotides (see FIG. 37 b). The PS-ODNrandomer (REP 2006) was significantly more efficacious against HSV-1than AC20 (REP 2055), TC20 (REP 2056) or AG20 (REP 2057) with theirefficacies ranked as follows: N40>AG>AC>TC. This data suggests thatalthough the anti-viral effect is non-sequence complementary, certainnon-specific sequence compositions (ie C40 and N40) have the most potentanti-viral activity. We suggest that this phenomenon can be explained bythe fact that, while retaining intrinsic protein binding ability,sequences like C40, A40, T40 and G40 bind fewer viral proteins with highaffinity, probably due to some restrictive tertiary structure formed inthese sequences. On the other hand, due to the random nature of N40, itretains its ability to bind with high affinity to a broad range ofanti-viral proteins which contributes to its robust anti-viral activity.

Example 3 Inhibition of CMV

The ability of PS-ODNs to inhibit CMV is measured in a plaque reductionassay (PRA). This assay is identical to the assay used for testinganti-HSV-1 and anti-HSV-2 except that CMV (strain AD169) is used as theviral innoculum and human fibroblasts were used as cellular host.

To test the size dependence and sequence independence of PS-ODNs onanti-CMV activity, we test PS-ODN randomers that vary in size (see FIG.16 a, b). When these PS-ODNs are tested in the CMV PRA, we find that asthe size of the PS-ODN increases, the potency also increases (IC₅₀decreases, see FIG. 16 c).

To more clearly elucidate the effective size range for PS-ODN anti-CMVactivity, we tested PS-ODN randomers covering a broader range of sizesfrom 10 to 80 bases. We also included several clinically accepted smallmolecule CMV treatments (Gancyclovir, Foscarnet and Cidofovir) as wellas 2 versions of a marketed antisense treatment for CMV retinitis,(Vitravene™; commercially available and synthesized by the University ofCalgary) (see FIG. 17). We discovered that while increased PS-ODNrandomer size leads to increased efficacy, this effect saturates at 40bases (see FIG. 18). Moreover, the 20, 40 and 80 base PS-ODN randomersare all significantly more efficacious than any of the small moleculetreatments tested (FIG. 17). In addition, 40 and 80 base PS-ODNrandomers are more efficacious than Vitravene™.

To the best of our knowledge, this is the first time an IC50 for CMV aslow as 0.067 μM has been reported for a PS-ODN.

Example 4 Inhibition of HIV-1

The ability of PS-ODN randomers to inhibit HIV-1 is measured by twodifferent assays:

Cytopathic Effect (CPE)

Cytopathic effect is monitored using MTT dye to report the extent ofcellular metabolism. Immortalized human lymphocyte (MT4) cells arecultured at 37° C. and 5% CO₂ in MEM plus 10% fetal calf serumsupplemented with antibiotics. Cells are seeded in 96 well plates inmedia containing the appropriate test compound and incubated for 2hours. After preincubation with the test compound, HIV-1 (strain NL 4-3)was added to the wells (0.0002 TCID₅₀/cell). After 6 days of additionalincubation, CPE is monitored by MTT conversion. Cytotoxicity is measuredby incubating the drugs for 6 days in the absence of viral inoculation.For transformation of MTT absorbance values into % survival, theabsorbance of uninfected, untreated cells is set to 100% and theabsorbance of infected, untreated cells is set to 0%.

Replication Assay (RA)

The ability of HIV to replicate is monitored in immortalized humanembryonic kidney (293A) cells. These cells are cotransfected with twoplasmids. One plasmid contains a recombinant wild type HIV-1 genome (NL4-3) having its env gene disrupted by a luciferase expression cassette(identified as strain CNDO), the other plasmid contains the env genefrom murine leukemia virus (MLV). These two plasmids provide all theprotein factors in trans to produce a mature chimeric virus having allthe components from HIV-1 except the protein products provided in transfrom the MLV env gene. Virions produced from these cells are infectiousand replicative but cannot produce another generation of infectiousvirions because they will lack the env gene products.

24 hours after transfection, these cells are trypsinized and plated in96 well plates. After the cells have adhered, the media is washed andreplaced with media containing the test compound. Virus production isallowed to proceed for an additional 24 hours. The supernatant is thenharvested and used to reinfect naive 293A cells. Naive cells that areinfected are identified by the luciferase gene product. The number ofluciferase positive cells is a measure of the extent of replicationand/or infection by the recombinant HIV-1. This assay is also adapted totest the resistance to many clinically accepted anti-HIV-1 drugs byusing a HIV-1 genome with several point mutations known to induceresistance to several different classes of anti-HIV drugs. Percentageinhibition is set to 100% for no detectable luciferase positive cellsand 0% for the number of positive cells in infected, untreated controls.

To test the size dependence and sequence independence of PS-ODNs onanti-HIV-1 activity, we test PS-ODN randomers that vary in size. Whenthese PS-ODN randomers are tested the HIV-1 CPE assay, we find that asthe size of the PS-ODN increases the potency also increases (IC₅₀decreases, see FIGS. 19 a, b and 20). We also noted that the PS-ODNrandomers exhibited no significant toxicity to the host cells in thisassay (see FIG. 19 c,d).

To the best of our knowledge, this is the first time an IC50 for HIV-1as low as 0.011 μM has been reported for a PS-ODN.

To more clearly elucidate the effective size range for PS-ODN anti-HIV-1activity, we tested more PS-ODN randomers covering a broader range ofsizes from 10 to 80 bases by RA using wild-type HIV-I (recombinant NL4-3 (CNDO)). In addition, we tested four protease inhibitors currentlyused in the clinic (aprenavir, indinavir, lopinavir and saquinavir). Wediscovered that PS-ODN randomers 10 bases and larger have anti-HIV-1activity and that the efficacy against HIV-1 also increases withincreased PS-ODN randomer length but is saturated at 40 bases (see FIG.21 e-h and FIG. 22 b). Moreover, the 40 and 80 base PS-ODN randomerswere almost equivalent in efficacy with the 4 clinical controls (seeFIG. 21 a-d and 22 a).

To the best of our knowledge, this is the first time an IC50 for HIV-1as low as 0.014 μM has been reported for a PS-ODN.

To test the ability of PS-ODN randomers to inhibit a drug resistantstrain of HIV, we duplicated the above test using the recombinant MDRC4strain of HIV-1. This recombinant strain exhibits significant resistanceto at least 16 different clinically accepted drugs from all classes:nucleotide RT inhibitors, non-nucleotide RT inhibitors and proteaseinhibitors. All the PS-ODN randomers perform as well against theresistant strain as they do against the wild type strain (see FIG. 23e-h). However, three of the four protease inhibitors show a reduction intheir efficacy against the mutant strain (see FIG. 23 a-d and 24), suchthat the 40 and 80 base PS-ODN randomers are now more potent againstthis resistant strain than these drugs.

Example 5 Inhibition of RSV

The ability of PS-ODN randomers to inhibit RSV is measured by monitoringCPE with alamar blue (an indirect measure of cellular metabolism). Humanlarynx carcinoma (Hep2) cells are cultured at 37° C. and 5% CO₂ in MEMplus 5% fetal calf serum. Cells are seeded in 96 well plates at adensity which yields a confluent monolayer of cells after 5-6 days ofgrowth. The day after plating, cells were infected with RSV (strain A2,10^(8.2) TCID₅₀/ml) in the presence of the test compound in a reducedvolume for 2 hours. Following inoculation, the media was changed and wassupplemented with test compound. 6 days after infection, CPE wasmonitored by measuring the fluorescent conversion of alamar blue.Toxicity of test compounds in Hep2 cells was monitored by treatinguninfected cells for 7 days and measuring alamar blue conversion inthese cells. The alamar blue readings in uninfected, untreated cellswere set to 100% survival and the readings in infected, untreated cellswere set to 0% survival.

To confirm the size dependence and sequence independence of PS-ODNs onanti-RSV activity, we test PS-ODN randomers that vary in size. Inaddition, we tested the clinically accepted treatment for RSV infection,Ribavirin (Virazole™). When tested in the RSV CPE assay, we find that asthe size of the PS-ODN randomer increases, the potency also increasesbut saturates at 40 bases in size (see FIG. 25 a-c and 26). We alsonoted that 20, 40 and 80 base PS-ODN randomers had IC₅₀ valuessignificantly lower than a clinically accepted anti-RSV drug, Ribavirin(see FIG. 25 a-d and 26). PS-ODN randomers exhibited no toxicity in Hep2cells while Ribavirin was significantly toxic (therapeutic index=2.08,see FIG. 25 e-h).

To the best of our knowledge, this is the first time an IC50 for RSV-1as low as 0.015 μM has been reported for a PS-ODN.

Example 6 Inhibition of Coxsackie Virus B2

The ability of PS-ODN randomers to inhibit COX B2 is measured monitoringCPE with alamar blue (an indirect measure of cellular metabolism).Rhesus monkey kidney (LLC-MK2) cells are cultured at 37° C. and 5% CO₂in MEM plus 5% fetal calf serum. Cells are seeded in 96 well plates at adensity which yields a confluent monolayer of cells after 5-6 days ofgrowth. The day after plating, cells were infected with COX B2 (strainOhio-1, 10^(7.8) TCID₅₀/ml) in the presence of the test compound in areduced volume for 2 hours. Following inoculation, the media was changedand was supplemented with test compound. 6 days after infection, CPE wasmonitored by measuring the fluorescent conversion of alamar blue.Toxicity of test compounds in LLC-MK2 cells was monitored by treatinguninfected cells for 7 days and measuring alamar blue conversion inthese cells. The alamar blue readings in uninfected, untreated cellswere set to 100% survival and the readings in infected, untreated cellswere set to 0% survival.

We tested the anti-COX B2 activity of REP 2006 in the COX B2 CPE assay.We found that, while exhibiting some slight toxicity in LLC-MK2 cells(see FIG. 27 b), this PS-ODN randomer was able to partially rescueinfected LLC-MK2 cells from COX B2 infection (see FIG. 27 a).

Example 7 Inhibition of Vaccinia Virus

We used the vaccinia infection model as a measure of the potentialefficacy of our compounds against poxviruses, including smallpox virus.The ability of PS-ODN randomers to inhibit Vaccinia is measured bymonitoring CPE with alamar blue (an indirect measure of cellularmetabolism). Vero cells are cultured at 37° C. and 5% CO₂ in MEM plus 5%fetal calf serum. Cells are seeded in 96 well plates at a density whichyields a confluent monolayer of cells after 5-6 days of growth. The dayafter plating, cells were infected with Vaccinia (10^(7.9) TCID₅₀/ml) inthe presence of the test compound in a reduced volume for 2 hours.Following inoculation, the media was changed and was supplemented withtest compound (all at 10 μM, except for Cidofovir which was used at 50μM). Five days after infection, the supernatants were harvested. Theviral load in the supernatant was determined by reinfection of VEROcells with supernatant diluted 1:100 and the monitoring of CPE 7 daysafter reinfection by measuring the fluorescent conversion of alamarblue.

We tested PS-ODN randomers that vary in size (REP 2004, 2006 and 2007).In addition, we tested a known effective treatment for Vacciniainfection, Cidofovir (Vistide™). When tested in the Vacinnia CPE assay,we find that treatment with REP 2004, 2006 and 2007 all displayedantiviral activity (ie. resulted in supernatants which showed adecreased CPE upon reinfection) but that this activity was weaker thanthat seen for Cidofovir (see FIG. 36).

Example 8 Inhibition of DHBV, Parainfluenza-3 Virus, and Hanta Virus

Because DHBV, Parainfluenza-3 virus and Hanta virus do not readilygenerate measurable plaques or CPE, we tested the efficacy of REP 2006in these viruses using a fluorescence focus forming unit (FFFU)detection. In this assay, REP 2006 (at a final concentration of 10 uM)is mixed with the virus which is then adsorbed onto the cells. Afteradsorption, infected cells are allowed to incubate for an additional7-14 days at which point they are fixed in methanol. Regions of viralreplication are detected by immunofluorescence microscopy against theappropriate viral antigen. For each of the three viruses tested, thespecific experimental conditions and results are described below: FFFUcount Antibody for FFFU count (10 uM REP Virus Cellular Host FFFUdetection (no drug) 2006) DHBV (HBV Primary duck Mouse anti-DHBV  163+/− 38.5 0 surrogate) hepatocytes IgG Parainfluenza-3 LLC-MK2 Mouseanti-PI3 288 +/− 26  0 cells IgG Hanta Virus VERO E6 Mouse anti- 232.3+/− 38.17 0 (Strain cells SinNombre Prospect Hill) nucleoprotein IgG

This initial data shows that at 10 uM, REP 2006 is effective ininhibiting DHBV, Parainfluenza-2 and Hanta Virus. We anticipate thatgiven the robust response in the preliminary test that IC₅₀ values willbe lower. These data support the efficacy of PS-ODN randomers for thetreatment of human infections of Hanta Virus and Hepatitis B (closelyrelated to DHBV) as well as providing a rationale for the immediatetreatment of pediatric bronchiolitis caused by RSV and Parainfluenza-3,which may not require differential diagnosis for treatment to begin.

Example 9 Currently Non-Responsive Viruses

To date we have not observed a detectable anti-viral efficacy withPS-ODN randomers (up to 10 uM) without using a delivery system, a drugcombination, or a chemical modification in the following viral systems:Assay Virus Strain Cellular Host paradigm Influenza A H3N2 MDCK cellsPlaque reduction Corona virus MHV2 (mouse) NCTC-1496 cells Plaque (SARSsurrogate) MHV-A59 (mouse) DBT cells reduction HCoV-OC43 HRT-18 cells(human) BVDV (HCV NA BT cells CPE by surrogate) alamar blue RhinovirusHGP HeLa cells CPE by alamar blue Adenovirus Human Ad5 293A cells Plaquereduction

Under the current testing procedures, we did not demonstrate activity.However, the lack of antiviral activity may be due to a lower cellularpenetration of the PS-ODN under the conditions of the assays.Nonetheless, additional testing is underway to achieve efficaciousresults with these viruses. These viruses may respond to PS-ODN whenusing a delivery system such as a liposomal formulation, in order toincrease its intracellular concentration. Also in a combination withanother antiviral drug, such as described herein, PS-ODNs may exhibit anantiviral efficacy for these viruses. A chemical modification toincrease intracellular concentration may also be useful to renderPS-ODNs active against these viruses.

Since we have good evidence that the charge characteristics of a PS-ODNare important for the inhibition of viruses from several differentfamilies, we expect that this charge dependent mechanism for theinhibition of viral activity has the potential to inhibit the activityof all encapsidating viruses. The corollary to this is that the lack ofdetected anti-viral efficacy against those viruses listed in Example 9suggests that the interaction between the PS-ODN and the structuralproteins of these viruses is not strong enough to prevent theinteraction of viral proteins during the replication of these viruses.One way of achieving efficacy against these viruses is to alter thecharge characteristics of the DNA or anti-viral polymer (e.g.,substituting phosphorbdithioate for phosphorothioate linkages in DNA) sotheir affinity for viral proteins is increased.

Example 10 Tests for Determining if an Oligonucleotide ActsPredominantly by a Non-Sequence Complementary Mode of Action

An ON, e.g., ODN, in question shall be considered to be actingpredominantly by a non-sequence complementary mode of action if it meetsthe criterion of any one of the 3 tests outlined below.

TEST #1—Effect of Partial Degeneracy on Antiviral Efficacy

This test serves to measure the antiviral activity of a particular ONsequence when part of its sequence is made degenerate. If the degenerateversion of the ON having the same chemistry retains its activity asdescribed below, is it deemed to be acting predominantly by anon-sequence complementary mode of action. ONs will be made degenerateaccording to the following rule:

-   -   L_(ON)=the number of bases in the original ON    -   X=the number of bases on each end of the oligo to be made        degenerate (but having the same chemistry as the original ON)    -   If L_(ON) is even, then X=L_(ON)/4    -   If L_(ON) is odd, then X=integer (L_(ON)/4)+1

Each degenerate base shall be synthesized according to any suitablemethodology, e.g., the methodology described herein for the synthesis ofPS-ODN randomers.

If the ON is claimed to have an anti-viral activity against a member ofthe herpesviridae, retroviridae, or paramyxoviridae families, the IC₅₀generation will be performed using the assay described herein for thatviral family preferably using the viral strains indicated. If the ON isclaimed to have an anti-viral activity against a member of a particularvirus family not mentioned above, then the IC50 values shall begenerated by a test of antiviral efficacy accepted by the pharmaceuticalindustry. IC50 values shall be generated using a minimum of sevenconcentrations of compound, with three or more points in the linearrange of the dose response curve. Using this test, the IC₅₀ of said ONshall be compared to its degenerate counterpart. If the IC₅₀ of thedegenerate ON is less than 2-fold greater than the original ON for an ONof 25 bases and less, or is less than 10-fold greater than the originalON for ONs 26 bases or more (based on minimum triplicate measurements,standard deviation not to exceed 15% of mean) then the ON shall bedeemed to be functioning predominantly by a non-sequence complementarymode of action.

TEST #2—Comparison of Efficacy with Randomer

This test serves to compare the anti-viral efficacy of an ON with theantiviral efficacy of a randomer ON of equivalent size and the samechemistry in the same virus or viral family.

If the ON is claimed to have an anti-viral activity against a member ofthe herpesviridae, retroviridae, or paramyxoviridae families, the IC₅₀generation will be performed using the assay described herein for thatviral family preferably using the viral strains indicated. If the ON isclaimed to have an anti-viral activity against a member of a particularvirus family not mentioned above, then the IC50 values shall begenerated by a test of antiviral efficacy accepted by the pharmaceuticalindustry. IC50 values shall be generated using a minimum of sevenconcentrations of compound, with three or more points in the linearrange of the dose response curve. Using this test, the IC₅₀ of the ONshall be compared to an ON randomer of equivalent size and the samechemistry. If the IC₅₀ of the degenerate ON is less than 2-fold greaterthan the original ON for an ON of 25 bases and less, or is less than10-fold greater than the original ON for ONs 26 bases or more (based onminimum triplicate measurements, standard deviation not to exceed 15% ofmean) then the ON shall be deemed to be functioning predominantly by anon-sequence complementary mode of action.

TEST #3—Comparison of Efficacy in a Different Viral Family

This test serves to compare the efficacy of an ON against a target viruswhose genome is homologous to the ON with the efficacy of the ON againsta second virus whose genome has no homology to ON. In many cases, thedifferent virus will be selected from a different viral family than theviral family of the target virus. The comparison of the relativeactivities of the ON in the target virus and the second virus isaccomplished by using the activities of a randomer of the same lengthand chemistry in the both viruses to normalize the IC₅₀ values for theON obtained in the two viruses.

Thus, if the ON is claimed to have an anti-viral activity against acertain virus, then the IC₅₀ generation will be determined in this virususing one of the assays described herein for the herpesviridae,retroviridae; or paramyxoviridae families, or other assay known in theart. Similarly, IC₅₀ generation will be performed for the ON against asecond virus using one of the assays as described herein for a viruswhose genome has no homology to the sequence of the ON. IC₅₀ generationis also performed for a randomer of equivalent size and chemistryagainst each of the viruses. The IC₅₀ efficacies of the randomer againstthe two viruses are used to normalize the IC₅₀ values for the specificON as follows:

-   -   1. An algebraic transformation is applied to the IC₅₀ of the ON        and the randomer in the first (homologous) virus such that the        IC₅₀ of the randomer is now 1.    -   2. An algebraic transformation is applied to the IC₅₀ of the ON        and the randomer in the second (non-homologous) virus such that        the IC₅₀ of the randomer in now 1.    -   3. The fold difference in the IC50s for the ON in the homologus        versus the non-homologous virus is calculated by dividing the        transformed IC50 of the ON in the non-homologous virus by the        transformed IC50 of the ON in the homologous virus.

For an ON less than 25 bases in length, the ON shall be deemed to beacting by a non-sequence complementary mode of action if the folddifference is less than 2. For an ON 25 bases or more in length, the ONshall be deemed to be acting by a non-sequence complimentary mode ofaction if the fold difference in less than 10.

TEST #4: Efficacy in a Different Viral Family

This test serves to determine if an ON has a drug like activity in avirus where the sequence of said ON is not homologous to any portion ofthe viral genome. Thus the ON shall be tested using one of the assaysdescribed herein for the herpesviridae, retroviridae or paramyxoviridaesuch that the sequence of the ON tested is not homologous to any portionof the genome of the virus to be used. An IC₅₀ value shall be generatedusing a minimum of seven concentrations of the ON, with three or morepoints in the linear range. If the resulting dose response curveindicates a drug like activity (which can be typically be seen as adecay or sigmoidal curve, having reduced anti-viral efficacy withdecreasing concentrations of ON) and the IC₅₀ generated from said curveis less than 1 uM, the ON shall be deemed to have a drug like activity.If the ON is deemed to have a drug like activity in a virus to which theON is not complementary and thus can have no complementary sequencedependent activity, it shall be considered to be acting by anon-sequence complementary mode of action.

Thresholds Used in these Tests

There is no scientific or empirical basis, either in the academic orindustrial fields, to design an antisense ON which is longer than 25bases. In addition, to our knowledge, there has never been an ONformulation administered in any human trials that used an ON longer than25 nucleotides.

Given these facts, we have established two different thresholds which weuse to define a sequence as acting predominantly by a non-sequencecomplimentary mode of action. For oligos 25 bases and less, the ON musthave an antiviral activity which is at least 2-fold greater than arandomer or a degenerate ON of the same chemistry in order to beconsidered to be acting predominantly by an antisense mechanism. If anantisense ON is not at least twice as good as a randomer or a degenerateON, then we conclude that more than half of its activity can beattributed to a non-antisense mode of action.

For ONs 26 bases and larger, the ON must have an antiviral activitywhich is at least 10-fold (1 log) greater than a randomer or adegenerate ON of the same chemistry to be considered to be acting by anantisense mechanism. Our rationale for this larger threshold it that,given the current state of the art for ON design for antisense, it isreasonable to assume that oligos that are 26 bases and larger andclaimed to have an antiviral activity were designed with the knowledgeof the invention contained herein (the optimal length of antisense ONsis generally accepted to be between 16 and 21 bases).

The thresholds described in tests 1 to 3 above are the defaultthresholds. If specifically indicated, other thresholds can be used inthe comparison tests 1 to 3 described above. Thus for example, for ONsunder 25 bases in length and/or ONs 25 or more bases in length, ifspecifically indicated the threshold for determining whether an ON isacting principally by a non-sequence complementary mode of action can beany of 10-fold, 8-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold,1.5-fold, or equal. The threshold described in test 4 above is also adefault threshold. If specifically indicated, the threshold fordetermining whether an ON is acting principally by a non-sequencecomplementary mode of action in test 4 can be an IC₅₀ of less than 1 uM,0.8 uM, 0.6 uM, 0.5 uM, 0.4 uM, 0.3 uM, 0.2 uM or 0.1 uM. Similarly,though the default is that satisfying any one of the above 4 tests issufficient, if specifically indicated, the ON can be required to satisfyany two (e.g., tests 1 & 2, 1 & 3, 1 & 4, 2&3, 2&4 and 3&4) or any three(e.g., tests 1 &2&3, 1 &3&4, and 2&3 & 4) or all 4 of the tests at adefault threshold, or if specifically indicated, at another threshold asindicated above.

Antiviral Assay for Herpesviridae

A plaque reduction assay performed as follows:

For HSV-1 or HSV-2, VERO cells (ATCC# CCL-81) are grown to confluence in12 well tissue culture plates (NUNC or equivalent) at 37 deg C. and 5%CO₂ in the presence of MEM supplemented with 10% heat inactivated fetalcalf serum and gentamycin, vancomycin and amphoterecin B. Upon reachingconfluency, the media is changed to contain 5% fetal calf serum andantibiotics as detailed above supplemented with either HSV-1 (strainKOS, 40-60 PFU total) or HSV-2 (strain MS2, 40-60 PFU total). Viraladsorbtion proceeds for 90 minutes, after which cells are washed andreplaced with new “overlay” media containing 5% fetal calf serum and 1%human imunoglobins. Three to four days after adsorbtion, cells are fixedby formalin and plaques are counted following formalin fixation.

For CMV, human fibroblasts are grown as specified for VERO cells in theHSV-1/2 assay. Media components and adsorbtion/overlay procedures areidentical with the following exceptions:

-   -   1. CMV (strain AD169, 40-60 PFU total) is used to infect cells        during the adsorbtion.    -   2. In the overlay media, 1% human immunoglobins are replaced by        4% sea-plaque agarose.

For other herpesviridae, testing is to be conducted in a plaque assaydescribed above using an appropriate cellular host and 40-60 PFU ofvirus during the adsorbtion.

This test is only valid if identifiable plaques are present in theabsence of compound at the end of the test.

IC₅₀ is the concentration at which 50% of the plaques are presentcompared to the untreated control.

Compound to be tested is present during the adsorption and in theoverlay.

Antiviral Assay for Retroviridae

Detection of total p24 in the supernatant of HIV-1 infected cells isperformed as follows:

Human PBMCs are infected with a primary isolate of HIV-1 in the presencethe compound. The cells are then incubated for an additional 7 days infresh medium supplemented with the compound after which the levels ofp24 in the supernatant are measured using a commercial p24 ELISA kit(BIOMERIEUX or equivalent.)

This test is only valid if there is an accumulation of p24 in the tissueculture supernatant in the infected, untreated cells.

IC₅₀ is the concentration at which the amount of p24 detectable is 50%of the p24 present in the untreated control.

Compound to be tested is present during the adsorption and in the mediaafter adsorption.

Antiviral Assay for Paramyxoviridae

For RSV, A measurement of CPE is performed as follows:

Hep2 cells were plated in 96 well plates and allowed to grow overnightin MEM plus 5% fetal calf serum at 37 deg C. and 5% CO₂. The next day,cells are infected with RSV (strain A2, 10^(8.2) TCID₅₀/ml in 100ul/well) by adsorbtion for 2 hours. Following adsorbtion, media ischanged and after 7 days growth, CPE is measured by conversion of AlamarBlue dye to its fluorescent adduct by living cells.

This test is only valid if CPE measurement (as measured by Alamar Blueconversion) in infected cells in the absence of compound is 10% of theconversion measured in uninfected cells.

For purposes of IC₅₀ comparison, 100% CPE is set at the conversion levelseen in infected cells and 0% CPE is set at the conversion seen inuninfected cells. Therefore IC₅₀ is the concentration of compound whichgenerates 50% CPE.

Compound to be tested is present during the adsorption and in the mediaafter adsorption.

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Forexample, variations can be made to synthesis conditions and compositionsof the oligonucleotides. Thus, such additional embodiments are withinthe scope of the present invention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numberical valuesare provided for embodiments, additional embodiments are described bytaking any 2 different values as the endpoints of a range. Such rangesare also within the scope of the described invention.

Thus, additional embodiments are within the scope of the invention andwithin the following claims. TABLE 1 DESCRIPTION OF OLIGONUCLEOTIDES REP1001 20 mer from human automonously replicating sequence SEQUENCE PS

REP 2001 22 mer from HSV-1 origin of replication SEQUENCE PS

REP 3007 16 mer from pUC19/pBR322 origin of replication SEQUENCE PS

REP 2002 5 mer randomer SEQUENCE PS

REP 2032 6 mer randomer SEQUENCE PS

REP 2003 10 mer randomer SEQUENCE PS

REP 2009 12 mer randomer SEQUENCE PS

REP 2010 14 mer randomer SEQUENCE PS

REP 2011 16 mer randomer SEQUENCE PS

REP 2012 18 mer randomer SEQUENCE PS

REP 2004 20 mer randomer SEQUENCE PS

REP 2006 30 mer randomer SEQUENCE PS

REP 2006 40 mer randomer SEQUENCE PS

REP 2007 80 mer randomer SEQUENCE PS

SEQUENCE PS

REP 2008 120 mer randomer SEQUENCE PS

SEQUENCE PS

REP 2013 10 mer randomer SEQUENCE N N N N N N N N N N no modificationREP 2014 20 mer randomer SEQUENCE N N N N N N N N N N N N N N N N N N NN no modification REP 2015 40 mer randomer SEQUENCE N N N N N N N N N NN N N N N N N N N N N N N N N N N N N N N N N N N N N N N N nomodifcation REP 2016 10 mer random sequence SEQUENCE PS

REP 2017 20 mer random sequence SEQUENCE PS

REP 2018 40 mer random sequence SEQUENCE PS

REP 2019 10 mer sequence centered around start codon of HSV-1 IE110protein (NCBI accession # X04614) SEQUENCE PS

REP 2020 20 mer sequence centered around start codon of HSV-1 IE110protein (NCBI accession # X04614) SEQUENCE PS

REP 2021 40 mer sequence centered around start codon of HSV-1 IE110protein (NCBI accession # X04614) SEQUENCE PS

REP 2024 40 mer randomer SEQUENCE PS 2-O Me

REP 2026 40 mer randomer SEQUENCE PCH3

REP 2036 21 mer commercially marketed antisense against CMV(vitravine/fomvirisen) SYNTHESIZED INTERNALLY SEQUENCE PS

REP 2036 © 21 mer commercially marketed antisense against CMV(vitravine/fomvirisen) COMMERCIAL PRODUCT (cGMP) SEQUENCE PS

A20 20 mer SEQUENCE PS

G20 20 mer SEQUENCE PS

C20 20 mer SEQUENCE PS

T20 20 mer SEQUENCE PS

AC10 20 mer SEQUENCE PS

AG10 20 mer SEQUENCE PS

TC10 20 mer SEQUENCE PS

TG10 20 mer SEQUENCE PS

REP 2029 40 mer SEQUENCE PS

REP 2028 40 mer SEQUENCE PS

REP 2031 40 mer SEQUENCE PS

REP 2030 40 mer SEQUENCE PS

REP 2055 40 mer SEQUENCE PS

REP 2056 40 mer SEQUENCE PS

REP 2057 40 mer SEQUENCE PS

REP 2059 20 mer RNA randomer SEQUENCE PS

REP 2060 30 mer RNA randomer SEQUENCE PS

1. An antiviral oligonucleotide formulation comprising at least oneantiviral oligonucleotide, wherein the antiviral activity of saidoligonucleotide occurs principally by a non-sequence complementary modeof action.
 2. An antiviral oligonucleotide formulation, comprising atleast one antiviral randomer oligonucleotide, wherein the antiviralactivity of said formulation occurs principally by a non-sequencecomplementary mode of action.
 3. An oligonucleotide formulation havingantiviral activity against a target virus, comprising at least oneantiviral oligonucleotide, wherein said oligonucleotide is at least 29nucleotides in length and the sequence of said oligonucleotide is notcomplementary to any portion of the genomic sequence of said targetvirus.
 4. An oligonucleotide formulation having antiviral activityagainst a target virus, comprising at least one antiviraloligonucleotide, wherein said oligonucleotide is at least 6 nucleotidesin length and the sequence of said oligonucleotide is not complementaryto a mRNA of said target virus and does not consist essentially ofpolyA, polyc, polyG, polyT, Gquartet, or a TG-rich sequence.
 5. Theoligonucleotide formulation of any of claims 1 to 4, wherein saidformulation has an IC₅₀ for a target virus of 0.05 μM or less.
 6. Theoligonucleotide formulation of any of claims 1 to 4, wherein saidoligonucleotide targets a DNA virus.
 7. The oligonucleotide formulationof any of claims 1 to 4, wherein said oligonucleotide targets a RNAvirus.
 8. The oligonucleotide formulation of any of claims 1 to 4,wherein said oligonucleotide is at least 29 nucleotides in length. 9.The oligonucleotide formulation of any of claims 1 to 4, wherein saidoligonucleotide is at least 40 nucleotides in length.
 10. Theoligonucleotide formulation of any of claims 1 to 4, wherein saidoligonucleotide comprises at least one modification to its chemicalstructure.
 11. The oligonucleotide formulation of any of claims 1 to 4,wherein said oligonucleotide is a concatemer consisting of two or moreoligonucleotide sequences joined by a linker.
 12. The oligonucleotideformulation of any of claims 1 to 4, wherein said oligonucleotide islinked or conjugated at one or more nucleotide residues, to a moleculemodifying the characteristics of the oligonucleotide to obtain one ormore characteristics selected from the group consisting of higherstability, lower serum interaction, higher cellular uptake, higher viralprotein interaction, an improved ability to be formulated for delivery,a detectable signal, higher antiviral activity, better pharmacokineticproperties, specific tissue distribution, lower toxicity.
 13. Theoligonucleotide formulation of any of claims 1 to 4, wherein saidoligonucleotide is double stranded.
 14. The oligonucleotide formulationof any of claims 1 to 4, wherein said formulation further comprises adelivery system.
 15. The oligonucleotide formulation of any of claims 1to 4, wherein said formulation further comprises a liposomalformulation.
 16. The oligonucleotide formulation of any of claims 1 to4, wherein said oligonucleotide comprises at least one Gquartet motifportion.
 17. The oligonucleotide formulation of any of claims 1 to 4,wherein said oligonucleotide comprises at least one CpG motif portion.18. The oligonucleotide formulation of any of claims 1 to 4, whereinsaid oligonucleotide binds to one or more viral components.
 19. Theoligonucleotide formulation of any of claims 1 to 4, wherein at least aportion of the sequence of said oligonucleotide is derived from a viralgenome.
 20. The oligonucleotide formulation of any of claims 1 and 4,comprising a mixture of at least two different antiviraloligonucleotides.
 21. An antiviral pharmaceutical composition comprisinga therapeutically effective amount of at least one pharmacologicallyacceptable, antiviral oligonucleotide according to any of claims 1 to 4,wherein the antiviral activity of said oligonucleotide occursprincipally by a non-sequence complementary mode of action; and apharmaceutically acceptable carrier.
 22. The antiviral pharmaceuticalcomposition of claim 21, adapted for delivery by a mode selected fromthe group consisting of intraocular injection, oral ingestion, enteralapplication, inhalation, topical application, subcutaneous injection,intramuscular injection, intraperitoneal injection, intrathecalinjection, intratrachael injection, and intravenous injection.
 23. Theantiviral pharmaceutical composition of claim 21, wherein saidcomposition further comprises a delivery system.
 24. The antiviralpharmaceutical composition of claim 21, wherein said composition furthercomprises a liposomal formulation.
 25. A kit comprising at least oneantiviral oligonucleotide or antiviral oligonucleotide formulationaccording to any of claims 1 to 4 in a labeled package, wherein theantiviral activity of said oligonucleotide occurs principally by anon-sequence complementary mode of action and the label on said packageindicates that said antiviral oligonucleotide can be used against atleast one virus.
 26. The kit of claim 25, wherein said kit is approvedby a regulatory agency for use in humans.
 27. A method for selecting anantiviral oligonucleotide for use as an antiviral agent, comprisingsynthesizing a plurality of different random oligonucleotides; testingsaid oligonucleotides for activity in inhibiting the ability of a virusto produce infectious virions; and selecting a said oligonucleotidehaving a pharmaceutically acceptable level of activity for use as anantiviral agent.
 28. A method for the prophylaxis or treatment of aviral infection in a subject, comprising administering to a subject inneed of such treatment a therapeutically effective amount of at leastone pharmacologically acceptable oligonucleotide according to any ofclaims 1 to
 4. 29. The method of claim 28, wherein said subject is ahuman.
 30. A method for the prophylactic treatment of cancer caused byoncoviruses in a human or a non-human animal, comprising administeringto a human or non-human animal in need of such treatment, apharmacologically acceptable, therapeutically effective amount of atleast one oligonucleotide according to any of claims 1 to
 4. 31. Themethod of claim 30, wherein said oligonucleotide is administered to ahuman.
 32. A method of screening to identify a compound that altersbinding of an oligonucleotide to at least one viral component, saidmethod comprising in separate reactions, contacting said oligonucleotidewith said viral component in the presence and absence of a compound tobe screened; and determining whether a difference occurs in binding ofsaid oligonucleotide to said viral component in the presence of saidcompound compared to in the absence of said compound, the presence ofsaid difference being indicative of said compound altering the bindingof said oligonucleotide to said viral component.
 33. A novel antiviralcompound identified by the method of claim
 32. 34. A method forpurifying oligonucleotides binding to at least one viral component froma pool of oligonucleotides comprising: contacting said pool with atleast one viral component; displacing bound oligonucleotides of saidpool from said viral component; and collecting displacedoligonucleotides.
 35. The method of claim 34, further comprisingsequencing, and testing antiviral activity of collected displacedoligonucleotides.
 36. A method for enriching oligonucleotides from apool of oligonucleotides binding to at least one viral component,comprising: contacting said pool with at least one viral component; andamplifying oligonucleotides bound to said viral component to provide anenriched oligonucleotide pool.
 37. The method of claim 36, wherein saidcontacting and amplifying are performed at least one additional timeusing said enriched oligonucleotide pool as the pool ofoligonucleotides.
 38. The method of claim 36, further comprisingsequencing and testing antiviral activity of oligonucleotides in saidenriched oligonucleotide pool.
 39. An antiviral oligonucleotidepreparation comprising one or more oligonucleotides identified using amethod of any of claim 34 or 36, wherein said oligonucleotides in saidoligonucleotide preparation exhibit higher mean binding affinity with atleast one viral component than the mean binding affinity ofoligonucleotides in the initial oligonucleotide pool.
 40. The antiviraloligonucleotide preparation of claim 39, wherein the mean bindingaffinity of said oligonucleotides is at least two-fold greater than themean binding affinity of oligonucleotides in the initial oligonucleotidepool.